AU2017336340B2 - Peronospora resistance in spinacia oleracea - Google Patents

Abstract

The present invention relates to an allele designated alpha-WOLF 15 which confers resistance to at least one Peronospora farinosa f. Sp. spinacea race, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif "MAEIGYSVC" at its N-terminus; and b) the motif "KWMCLR"; and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:10. The allele when homozygously present in a spinach plant confers complete resistance to Peronospora farinosa f. Sp. spinacea races pfs:1, pfs:2, pfs:3, pfs:4 and pfs: 5, pfs:6, pfs:8, pfs:9, pfs:11, pfs:12, pfs:13, pfs:14, pfs:15, pfs:16 and isolates UA1014 and US1508, and confers intermediate resistance to pfs:10, and does not confer resistance to pfs:7.

Description

PERONOSPORA RESISTANCE IN SPINACIA OLERACEA

FIELD OF THE INVENTION The invention relates to an allele capable of conferring resistance to a spinach plant

against multiple Peronosporafarinosa f. sp. spinaciaeraces. The invention also relates to a

spinach plant, to propagation material of said spinach plant, to a cell of said spinach plant, and to

seed of said spinach plant carrying the allele. The invention further relates to a method of

producing a spinach plant carrying the allele and to the use of the allele in breeding to confer

resistance against Peronosporafarinosaf. sp. Spinaciae.

BACKGROUND OF THE INVENTION Downy mildew (Peronosporafarinosa f. sp. spinaciae)is a major threat for spinach

growers because it directly affects the harvested leaves. In spinach, downy mildew is caused by the

oomycete Peronosporafarinosaf. sp. spinaciae (formerly known as P. effusa). Infection makes

the leaves unsuitable for sale and consumption, as it manifests itself phenotypically as yellow

lesions on the older leaves, and on the abaxial leaf surface a greyish fungal growth can be

observed. The infection can spread very rapidly, and it can occur both in glasshouse cultivation and

in soil cultivation. The optimal temperature for formation and germination of P.farinosaf. sp.

spinaciae spores is 9 to 12°C, and it is facilitated by a high relative humidity. When spores are

deposited on a humid leaf surface they can readily germinate and infect the leaf. Fungal growth is

optimal between 8 and 20°C and a relative humidity of >80%, and within 6 and 13 days after

infection mycelium growth can be observed. Oospores of P. farinosacan survive in the soil for up

to 3 years, or as mycelium in seeds or living plants.

To date 16 pathogenic races of spinach downy mildew (Pfs) have been officially

identified and characterized, and many new candidates are observed in the field. The 16 officially

recognised races of Peronosporafarinosaf. sp. spinaciae, are designated Pfs:1 to Pfs:16 (Irish et

al. Phtypathol. Vol. 98 pg. 894-900, 2008; Plantum NL (Dutch association for breeding, tissue culture, production and trade of seed and young plants) press release, "Benoeming van Pfs: 14, een

nieuwe fysio van valse meeldauw in spinazie", September 19, 2012; Report Jim Correl (Univ.

Arkansas) and Steven Koike (UC Cooperative Extension, Monterey County), "Race Pfs: 14

Another new race of the spinach downy mildew pathogen", September 18, 2012; Plantum NL press

release, "Denomination of Pfs: 15, a new race of downy mildew in spinach", September 2, 2014,

Plantum NL press release, "Denomination of Pfs: 16, a new race of downy mildew in spinach,

March 15, 2016). Races 4 to 15 were identified between 1990 and 2014, while only recently another new Peronosporaisolate has been identified, termed UA201519B, which subsequently has

been officially named Pfs:16 by the International Working Group on Peronospora (IWGP)

(Plantum NL (Dutch association for breeding, tissue culture, production and trade of seed and

young plants) press release, "Denomination of Pfs: 16, a new race of downy mildew in spinach",

March 15, 2016. All 16 officially recognized Pfs races are publicly available from the Department

of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA, and also from NAK Tuinbouw, Sotaweg 22, 2371 GD Roelofarendsveen, the Netherlands.

Especially the latest identified Peronosporaraces can break the resistance of many

spinach varieties that are currently used commercially worldwide, and they thus pose a serious

threat to the productivity of the spinach industry. Therefore, it is crucial to stay at the forefront of

developments in this field, as Peronosporacontinuously develops the ability to break the

resistances that are present in commercial spinach varieties. For this reason new resistance genes

against downy mildew are very valuable assets, and they form an important research focus in

breeding and particular in spinach and lettuce breeding. One of the main goals of spinach breeders

is to rapidly develop spinach varieties with a resistance to as many Peronosporaraces as possible,

including the latest identified races, before these races become wide-spread and pose a threat to

the industry.

In commercial spinach varieties resistance against downy mildew is usually caused by

so-called R-genes. R-gene mediated resistance is based on the ability of a plant to recognize the

invading pathogen. In many cases this recognition occurs after the pathogen has established the

first phases of interaction and transferred a so called pathogenicity (or avirulence) factor into the

plant cell. These pathogenicity factors interact with host components in order to establish

conditions which are favorable for the pathogen to invade the host and thereby cause disease.

When a plant is able to recognize the events triggered by the pathogenicity factors a resistance

response can be initiated. In many different plant pathogen interaction systems such as the

interaction of spinach with different downy mildew strains, the plant initiates these events only

after specific recognition of the invading pathogen.

Co-evolution of plant and pathogen has led to an arms race in which a R-gene

mediated resistance is sometimes overcome as a consequence of the capability of the pathogen to

interact with and modify alternative host targets or the same targets in a different way, such that

the recognition is lost and infection can be established successfully resulting in disease. In order to

re-establish resistance in a plant, a new R-gene has to be introduced which is able to recognize the

mode of action of an alternative pathogenicity factor.

Despite the fact that the durability of R-genes is relatively low, R-genes are in spinach

still the predominant form of defense against downy mildew. This is mainly due to the fact that it is

the only form of defense that gives absolute resistance. So far plant breeders have been very

successful in generating downy mildew resistant spinach varieties by making use of resistance

genes residing in the wild germplasm of the crop species. Even though R-genes are extensively used in spinach breeding, until now not much is known of these R-genes. The R-genes present in the current commercial spinach varieties have never been characterized at the molecular level, i.e. their sequence until now was unknown. Also up until now there are no closely linked molecular markers known in the art that separate these R-genes, nor are the molecular characteristics of the genes themselves known in the art. Therefore, the search for new R-genes and R-gene identification is currently based on phenotypic assays in which many accessions are screened for possible variation in their resistance pattern. Subsequently it has to be determined through crossing and selection whether a newly observed resistance is in fact caused by an R-gene. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". Adequately responding to newly emerging downy mildew races is crucial for developing commercially successful spinach varieties. In that regard, the present invention relates to a new resistance allele conferring resistance to a newly emerged downy mildew isolate and to provide molecular biological tools for identifying this new resistance allele. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION In one aspect, the present disclosure provides a method for identifying or selecting a spinach plant carrying an allele designated alpha-WOLF 15 which confers resistance to at least one Peronosporafarinosaf. Sp. spinacea race when present in a spinach plant, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif "MAEIGYSVC" at its N-terminus; and b) the motif "KWMCLR"; and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:10, and wherein the allele when homozygously present in a spinach plant confers complete resistance to Peronospora farinosa f. Sp. spinacea races pfs:1, pfs:2, pfs:3, pfs:4 and pfs: 5, pfs:6, pfs:8, pfs:9, pfs:11, pfs:12, pfs:13, pfs:14, pfs:15, pfs:16 and isolates UA1014 and US1508, and confers intermediate resistance to pfs:10, and does not confer resistance to pfs:7, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order

3a

of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:1. In another aspect, the present disclosure provides a method for identifying or selecting a spinach plant carrying the allele as described herein, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 9 7%, 99%, 9%, 100% sequence similarity to SEQ ID No:2. In another aspect, the present disclosure provides a method for identifying or selecting a spinach plant carrying an allele as described herein, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 9 7%, 99%, 9%, 100% sequence similarity to SEQ ID No:3. In another aspect, the present disclosure provides use of a primer pair comprising a forward primer which is a nucleic acid molecule having the sequence of SEQ ID No:6 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:7. In another aspect, the present disclosure provides a method for producing a spinach plant showing resistance to Peronosporafarinosaf. sp. spinaciae comprising: (a) crossing a plant comprising an allele as described herein, with another plant; (b) optionally performing one or more rounds of selfing and/or crossing; (c) selecting after one or more rounds of selfing and/or crossing for a plant that comprises an allele as described herein. In the research leading to the present invention, it was found that different resistance genes that confer resistance to Peronosporafarinosaf. sp. spinaciaein spinach are not separate resistance loci, as had been previously assumed, but that they are different alleles of the same one or two genes. These one or two genes, which are either "alpha-WOLF' type or "beta-WOLF' type genes (together referred to as "the WOLF genes") each encode a protein that belongs to the CC NBS-LRR family (Coiled Coil - Nucleotide Binding Site - Leucine-Rich Repeat). Depending on the allelic variant (or the allelic variants) that is (are) present in a spinach plant, said plant will produce a variant of the WOLF protein that confers a certain resistance profile to pathogenic races of Peronosporafarinosaf. sp. spinaciae. The research leading to the present invention has furthermore elucidated the relationship between the different alleles present in the genome of a spinach plant and the resistance profile of said plant to a number of different pathogenic races of Peronosporafarinosaf. sp. spinaciae. In the context of this invention the term "allele" or "allelic variant" is used to designate a version of the gene that is linked to a specific phenotype, i.e. resistance profile. It was found that a spinach plant may carry one or two WOLF genes. Each of these two WOLF genes encompasses multiple alleles, each allele conferring a particular resistance profile. The beta WOLF

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gene is located on scaffold12735 (sequence: GenBank: KQ143339.1), at position 213573-221884. In case the spinach plant also carries or only carries the alpha-WOLF gene, the alpha-WOLF gene is located at approximately the same location as where the beta-WOLF gene is located on scaffold12735 in the Viroflay genome assembly.

A screen for novel WOLF-alleles in the spinach germplasm identified a new allele of

the alpha-WOLF gene conferring a new and unique resistance profile against several downy

mildew races including the recently identified race pfs:16.

DETAILED DESCRIPTION OF THE INVENTION A genome assembly for spinach variety Viroflay - which is susceptible to all known

f. sp. spinaciae- is publicly available (Spinacia pathogenic races of Peronosporafarinosa

oleraceacultivar SynViroflay, whole genome shotgun sequencing project; Bioproject:

PRJNA41497; GenBank: AYZV00000000.2; BioSample: SAMN02182572, see also Dohm et al, 2014, Nature 505: 546-549). In this genome assembly for Viroflay, the beta-WOLF gene is located on scaffold12735 (sequence: GenBank: KQ143339.1), at position 213573-221884. The sequence covered by this interval comprises the entire genomic sequence of the beta-WOLF gene of

Viroflay, plus 2000 basepairs sequence upstream from the gene, plus the sequence downstream

from the gene, up to the locus of the neighbouring gene that is situated downstream from the

WOLF gene.. Spinach variety Viroflay only possesses a single WOLF gene, namely a beta-WOLF

gene, but most other spinach lines harbor a single alpha-type WOLF gene at the same location in

the genome. Other spinach lines harbor two WOLF genes at approximately the same location in the

genome. In such cases, the two WOLF genes are positioned adjacent to each other. In most spinach

lines that harbor two WOLF genes, one of said WOLF genes belongs to the alpha-type, and the

other WOLF gene belongs to the beta-type. In the research leading to the present invention, it was

observed that this allelic variation in the WOLF locus is responsible for differences in resistance to

pathogenic races of Peronosporafarinosa f. sp. spinaciae.

The difference between an allele of an alpha-WOLF gene and an allele of a beta

WOLF gene lies in the presence of specific conserved amino acid motifs in the encoded protein

sequence. As mentioned above, all WOLF proteins possess - from N- to C-terminus - the

following domains that are generally known in the art: a coiled coil domain (RX-CC-like,

cd14798), an NBS domain (also referred to as "NB-ARC domain", pfam00931; van der Biezen &

Jones, 1998, Curr. Biol. 8: R226-R228), and leucine-rich repeats (IPR032675) which encompass the LRR domain. In addition, all WOLF proteins comprise in their amino acid sequence the motif

"MAEIGYSVC" at the N-terminus. In addition to this, all alpha-WOLF proteins comprise the

motif "KWMCLR" in their amino acid sequence, whereas all beta-WOLF proteins comprise the

motif "HVGCVVDR" in their amino acid sequence.

The present invention relates to a new Peronosporafarinosaf. sp. spinaciaeresistance conferring

allele of the alpha-WOLF gene designated alpha-WOLF 15. In particular, the invention relates to a Peronosporafarinosaf. sp. spinaciae

resistance conferring allele designated alpha-WOLF 15 wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif "MAEIGYSVC" at its N-terminus; and b) the motif "KWMCLR"; and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:10. Optionally, the alpha-WOLF 15 allele further comprise an additional motif in their amino acid sequence, namely "DQEDEGEDN". For the purpose of this invention, the LRR domain of the protein of the alpha-WOLF 15 allele is defined as the amino acid sequence that in order of increased preference has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:10. The skilled person is familiar with methods for the calculation of sequence similarity. Suitably sequence similarity is calculated using EMBOSS stretcher 6.6.0 (wwwebi.ac~uJTools/ps-emboss stretcher), using the EBLOSUM62 matrix and the resulting "similarity score". The LRR domain of the alpha-WOLF 15 allele as defined herein can be determined by amplifying and sequencing the genomic DNA encoding for the amino acid sequence of LRR domain using specific primers, and subsequently translating the DNA sequence into an amino acid sequence, thereby applying common sense in choosing the correct reading frame. The skilled person is capable of doing this, using freely available online bioinformatics tools such as can be found here: it:I/webexas or/translate/ The genomic sequence of a LRR domain of an alpha-WOLF gene such as alpha WOLF 15 can be amplified using a primer pair having a forward primer which is a nucleic acid molecule having the sequence of SEQ ID No:6 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:7. PCR conditions for amplifying the LRR domain-encoding region of an alpha- WOLF gene using primers having SEQ ID No:6 and SEQ ID No:7 are, using Platinum Taq enzyme (Thermo Fisher Scientific): 3 minutes at 95°C (initial denaturing step); 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C, 30 seconds annealing at 60°C, and 30 seconds extension at 72°C; 2 minutes at 72°C (final extension step). The LRR domain of a beta-WOLF gene, e.g. the null allele as present in variety Viroflay, can be amplified using a forward primer which is a nucleic acid molecule having the sequence of SEQ ID No:8 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:7. PCR conditions for amplifying the LRR domain-encoding region of a beta- WOLF gene using primers having SEQ ID No:7 and SEQ ID No:8 are as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):- 3 minutes at 95°C (initial denaturing step); 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C, 50 seconds annealing at 58°C and 50 seconds extension at 72°C; 2 minutes at 72°C (final extension step).

Therefore, the invention also relates to a primer pair for amplifying the LRR domain of an alpha-WOLF gene, more in particular for amplifying the LRR domain of an alpha-WOLF 15 allele wherein the forward primer is a nucleic acid molecule having the sequence of SEQ ID No:6 and the reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:7. The primers disclosed herein have been specifically designed for selectively amplifying part of a WOLF gene, and not of any other CC-NBS-LRR protein-encoding genes. The invention relates to an alpha-WOLF 15 allele which has a genomic sequence that in order of increased perence has at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:1. o The invention relates to two different splice variants. In one embodiment, the invention relates to an alpha-WOLF 15 allele which has a coding sequence which in order of increased preference has at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:2. This is the first splice variant of the alpha-WOLF 15 allele. -5 In a further embodiment the alpha-WOLF 15 allele has a coding sequence which in order of increased perence has at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, 100% sequence similarity to SEQ ID No:3. This is the second splice variant. In a further aspect of the invention the alpha-WOLF 15 allele encodes for a protein o having an amino acid sequence which in order of increased preference has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:4. In another embodiment the alpha-WOLF 15 allele encodes for a protein having an amino acid sequence which in order of increased preference has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:5 (isoform 1). The alpha-WOLF 15 allele when homozygously present in a spinach plant confers complete resistance to the officially recognized Peronosporafarinosaf. Sp. spinacearaces pfs:1, pfs:2, pfs:3, pfs:4, pfs:5, pfs:6, pfs:8, pfs:9, pfs:11, pfs:12, pfs:13, pfs:14, pfs:15, UA1014 and US1508 and confers intermediate resistance to pfs:10 and does not confer resistance to downy mildew race pfs:7 (See Table 1). As indicated in Table 1, a spinach plant heterozygous for the alpha WOLF 15 allele and not carrying any other resistance conferring allele will be intermediately resistant for downy mildew races Pfs:8, and Pfs:10 and susceptible to Pfs:7. The resistance of a spinach plant against one or more races of Peronosporafarinosaf. sp. spinaciae can be determined using a seedling test. Herein, a seedling test is defined as a test wherein spinach plants are planted in trays containing growth medium, optionally fertilized twice a week after seedling emergence. Plants were inoculated at the first true leaf stage with a sporangial suspension having a concentration of approximately 2.5 x 105Iml of one of the pathogenic races of

Peronosporafarinosaf. sp. spinaciaeor isolates to be tested. The inoculated plants are placed in a

dew chamber at 18°C with 100% relative humidity for a 24 h period, and then moved to a growth

chamber at 18°C with a 12 h photoperiod for 6 days. After 6 days, the plants are returned to the

dew chamber for 24 h to induce sporulation, and subsequently scored for a disease reaction.

Preferably, 30 plants per race are tested.

As used herein, a plant is completely resistant against a Peronosporafarinosaf. sp.

spinaciae race when a plant shows no symptoms in the seedling test described herein.

As used herein, a plant is intermediately resistant against a Peronosporafarinosaf.

sp. spinaciaerace when a plant shows only symptoms of chlorosis, or sporulation occurring only

on the tips of the cotyledons in the seedling test described herein.

As used herein, a plant is susceptible to an isolate of a Peronosporafarinosaf. sp.

spinaciae race when a plant shows more than only symptoms of chlorosis, or when sporulation

occurs on area larger than only the tips of the cotyledons in the seedling test described herein.

Another aspect of the invention relates to a spinach plant, comprising the alpha

WOLF 15 allele of invention, of which a representative sample of seed was deposited with the

NCIMB under NCIMB accession number 42466. In a further embodiment the plant of the invention which comprises the alpha-WOLF

15 allele is an agronomically elite spinach plant.

In the context of this invention an agronomically elite spinach plant is a plant having a

genotype that results into an accumulation of distinguishable and desirable agronomic traits which

allow a producer to harvest a product of commercial significance, preferably the agronomically

elite spinach plant comprising the alpha-WOLF 15 allele is a plant of an inbred line or a hybrid.

As used herein, a plant of an inbred line is a plant of a population of plants that is the

result of three or more rounds of selfing, or backcrossing; or which plant is a double haploid. An

inbred line may e.g. be a parent line used for the production of a commercial hybrid.

As used herein, a hybrid plant is a plant which is the result of a cross between two

different plants having different genotypes. More in particular, a hybrid plant is the result of a cross

between plants of two different inbred lines, such a hybrid plant may e.g. be a plant of an F1 hybrid

variety. A plant carrying the alpha-WOLF 15 allele in heterozygous form may further

comprise a beta-WOLF 0 allele as e.g. present in variety Viroflay wherein the beta-WOLF 0 allele

does not confer any resistance to downy mildew. However, a plant heterozygous for the alpha

WOLF 15 allele may further comprise an allele of the alphabeta-WOLF gene that does provide

resistance to downy mildew. Preferably, such an allele would complement the alpha-WOLF 15

allele such that the spinach plant will be at least intermediately resistant to one or more other races to which the alpha-WOLF 15 allele does not provide resistance. Most preferably, the other allele of the alpha/beta-WOLF gene complements the alpha-WOLF 15 allele such that the plant is resistant to Peronosporafarinosaf. sp. spinaciaeraces Pfs:1 to Pfs:16. In one embodiment such a plant is an agronomically elite plant. Alternatively, the resistance profile of a plant carrying the alpha-WOLF 15 allele is complemented by a resistance conferring allele of a totally different gene. Examples of such genes are e.g. DMR1 as described in US8,354,570 and DMR6 as described in US9,121,029. The invention thus relates to a spinach plant carrying the alpha-WOLF 15 allele and further comprising a genetic determinant resulting in resistance against Peronosporafarinosaf. Sp. spinacea races pfs:1 to pfs:16. The genetic determinant can be another resistance conferring alphalbeta-WOLF allele or a resistance conferring allele of a totally different gene. The invention further relates to propagation material comprising the alpha-WOLF 15 allele. In one embodiment, the propagation material is suitable for sexual reproduction. Such propagation material comprises for example a microspore, pollen, ovary, ovule, embryo sac and egg cell. In another embodiment, the propagation material is suitable for vegetative reproduction. Such propagation material comprises for example a cutting, root, stem, cell, protoplast, and a tissue culture of regenerable cells. A part of the plant that is suitable for preparing tissue cultures is in particular a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root tip, an anther, a flower, a seed and a stem. The invention furthermore relates to a cell of a spinach plant comprising the alpha WOLF 15 allele. Such a cell may be either in isolated form or may be part of the complete plant or parts thereof and then still constitutes a cell of the invention because such a cell harbors the alpha WOLF 15 allele that confers resistance to downy mildew. Each cell of a plant of the invention carries the genetic information that confers resistance to Peronosporafarinosaf. sp. spinaciae .

Such a cell of the invention may also be a regenerable cell that can be used to regenerate a new plant comprising the allele of the invention. Yet another aspect of the invention relates to a method for making a hybrid spinach seed comprising crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first and/or second parent spinach plant comprises the alpha-WOLF 15 allele. In particular embodiment, the first and/or second parent plant is a plant of an inbred line as defined herein. The invention further relates hybrid spinach plant grown from seed produced by crossing a first parent spinach plant with a second parent spinach plant and harvesting the resultant hybrid spinach seed, wherein said first and/or second parent spinach plant comprises the alpha WOLF 15 allele.

Another aspect of the invention relates to a method for identifying or selecting a spinach plant carrying the alpha-WOLF 15 allele, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:1. The invention further relates to a method for identifying or selecting a spinach plant carrying the alpha-WOLF 15 allele, comprising determining the presence of a coding sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:2. The invention further relates to a method for identifying or selecting a spinach plant carrying the alpha-WOLF 15 allele , comprising determining the presence of a coding sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:3. Determining the genomic DNA or coding DNA sequence of at least part of a WOLF gene in the genome of a spinach plant may be performed using any suitable molecular biological method known in the art, including but not limited to (genomic) PCR amplification followed by Sanger sequencing, whole-genome-sequencing, transcriptome sequencing, sequence-specific target capture followed by next-generation sequencing (using, for example, the xGen* target capture system of Integrated DNA Technologies), specific amplification of LRR-domain-comprising gene sequences (using, for example, the RenSeq methodology, as described in US patent application 14/627116, and in Jupe et al., 2013, PlantJ. 76: 530-544) followed by sequencing, etcetera. In another embodiment the invention relates to a method for identifying or selecting a plant carrying the alpha-WOLF 15 allele comprises determining the DNA sequence coding for the LRR domain as defined herein. In a further embodiment of the method the LRR domain of the alpha-WOLF 15 allele is determined by using a primer pair to amplify the genomic DNA region of the LRR domain. The forward primer is preferably a nucleic acid molecule having the sequence of SEQ ID No:6 and the reverse primer is preferably a nucleic acid molecule having the sequence of SEQ ID No:7. Another aspect of the invention relates to a method for producing a spinach plant comprising resistance to Peronosporafarinosaf. sp. spinaciae comprising: (a) crossing a plant comprising the alpha-WOLF 15 allele, with another plant; (b) optionally performing one or more rounds of selfing and/or crossing; and (c) optionally selecting after each round of selfing or crossing for a plant that comprises the alpha-WOLF 15 allele. Selecting a plant comprising the alpha-WOLF 15 allele can be done genotypically by determining the presence of the genomic DNA sequence of the allele having in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:1. In another embodiment, selecting a plant comprising the alpha-WOLF 15 allele can be done genotypically by determining the presence the coding sequence of the allele having in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:2, In another embodiment, selecting a plant comprising the alpha-WOLF 15 allele can be done genotypically by determining the presence the coding sequence of the allele having in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%sequence similarity to SEQ ID No:3. Alternatively, the presence of the alpha-WOLF 15 allele can be determined phenotypically by assaying a plant in a disease test, for example the test as described herein, and identifying a plant carrying the alpha-WOLF 15 allele based on the resistance pattern as described herein and indicated in Table 1. The invention further relates to the use of a spinach plant carrying the alpha-WOLF

15 allele in breeding to confer resistance against Peronosporafarinosaf. sp. spinaciae.

The invention also relates to a breeding method for the development of spinach plants

carrying the alpha-WOLF 15 allele of the invention wherein germplasm which comprises said

allele is used. Seed capable of growing into a plant comprising the allele of the invention and being

representative for the germplasm was deposited with the NCIMB under deposit number NCIMB

42466. In another aspect, the invention relates to a method for the production of a spinach

plant which comprises alpha-WOLF 15 allele, which method comprises: (a) crossing a plant

comprising the allele with another plant; (b) optionally selecting for plants comprising said allele in

the F1; (c) optionally backcrossing the resulting F1 with the preferred parent and selecting for

plants that have the said allele in the BClF1; (d) optionally performing one or more additional

rounds of selfing, crossing, and/or backcrossing, and subsequently selecting for a plant which

comprises the said allele or shows the resistance profile corresponding to said allele. The invention

also encompasses a spinach plant produced by this method.

The invention also relates to a harvested leaf of a spinach plant of the invention, to a

food product which comprises a harvested leaf of a spinach plant of the invention, either in natural

or in processed form.

Spinach leaves are sold in packaged form, including without limitation as pre

packaged spinach leaves or as processed in a salad comprising said leaves. Mention of such a

package is e.g. made in US Patent No. 5,523,136, which provides packaging film, and packages from such packaging film, including such packaging containing leafy produce, and methods for making and using such packaging film and packages, which are suitable for use with the spinach leaves of the invention. Thus, the invention comprehends the use of and methods for making and using the leaves of the spinach plant of the invention, as well as leaves of spinach plants derived from the invention.

The invention further relates to a container which comprises one or more plants of the

invention, or one or more spinach plants derived from a plant of the invention, in a growth

substrate for harvest of leaves from the plant, in a domestic environment. This way the consumer

may pick very fresh leaves for use in salads, when the plant is in a ready-to-harvest condition.

The invention also relates to the use of a spinach plant, of which representative seed

was deposited with the NCIMB under accession number NCIMB 42466, in the production of a

spinach plant comprising the alpha-WOLF 15 allele. In a further embodiment the said spinach plant is a hybrid, doubled haploid, or inbred

spinach plant

Another aspect of the invention is the use of a cell comprising the alpha-WOLF 15

allele for the production of a spinach plant showing resistance to Peronosporafarinosaf. sp.

spinaciae.

The invention also relates to the use of a tissue culture comprising the alpha-WOLF

15 allele for the production of a spinach plant showing resistance to Peronosporafarinosa f. sp.

spinaciae.

RESISTANCE INFORMATION Table 1 Resistance profile conferred by the Alpha-WOLF 15 allele. A "-" means complete resistance

against a particular downy mildew race; "(-)" means intermediate resistance against a particular

downy mildew race; "+" means that the allele confers no resistance and would cause a plant only

carrying the Alpha-WOLF 15 allele to be fully susceptible for that particular downy mildew race. * The resistance against Pfs:8 as conferred by the alpha WOLF 15 allele is only observed in

homozygous state. A plant carrying the allele in heterozygous state and not carrying any other

resistance conferring allele (i.e. carrying the beta-WOLF zero allele) would be intermediate

resistant for Pfs:8.

Alpha-WOLF 15 resistance profile Peronosporafarinosa f. Resistance score Peronosporafarinosa f. Resistance score sp. Spinaciae race sp. Spinaciae race Pfs:1 Pfs:10 (-)

Pfs:2 - Pfs:ll Pfs:3 - Pfs:12 Pfs:4 - Pfs:13

Pfs:5 - Pfs:14 Pfs:6 - Pfs:15 Pfs:7 + Pfs:16

+ Pfs:8 * UA1014 Pfs:9 US1508

DEPOSIT INFORMATION Seeds of a plant comprising the alpha-WOLF 15 allele of the invention in its genome

were deposited with NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen

AB21 9YA, UK, on October 15, 2015, under deposit accession number 42466. The deposit was

made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon

the deposit will be removed, and the deposit is intended to meet the requirements of 37 CFR §

1.801-1.809. The deposit will be irrevocably and without restriction or condition released to the

public upon the issuance of a patent. The deposit will be maintained in the depository for a period

of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is

longer, and will be replaced if necessary during that period.

SEQUENCE INFORMATION Table 2 SEQ ID No:1: ATGGCCGAAATCGGATACTCGGTTTGTGCGAAACTCATCGAAGTG Genomic ATTGGCAGTGAGCTGATCAAAGAGATTTGTGACACATGGGGTTAC AAATCTCTTCTTGAGGACCTCAACAAAACTGTATTGACGGTCAGG sequence of AACGTTCTCATTCAAGCCGGGGTGATGCGGGAGCTTACTAGTGAA alpha-WOLF 15 CAACAAGGTTTCATTGCAGACCTTAAAGATGTTGTTTATGATGCTG ATGACTTGTTCGACAAGTTACTCACTCGTGCTGAGCGAAAACAGA TTGATGGAAACGAAATCTCTGAAAAGGTACGTCGTTTCTTTTCCTC TAGTAACAAGATCGGTCAAGCTTACTACATGTCTCGTAAGGTTAA GGAAATTAAGAAGCAGTTGGATGAAATTGTTGATAGGCATACAAA ATTTGGGTTTAGTGCCGAGTTTATACCTGTTTGTAGGGAAAGGGGG AACGAGAGGGAAACACGTTCATATATAGATGTCAAGAATATTCTT GGGAGGGATAAAGATAAGAATGATATCATAGATAGGTTGCTTAAT CGTAATGGTAATGAAGCTTGTAGTTTCCTGACCATAGTGGGAGCG GGAGGATTGGGAAAAACTGCTCTTGCACAACTTGTGTTCAATGAT GAAAGGGTCAAAATTGAGTTCCATGATTTGAGGTATTGGGTTTGT GTCTCTGATCAAGATGGGGGCCAATTTGATGTGAAAGAAATCCTT TGTAAGATTTTAGAGGTGGTTACTAAGGAGAAAGTTGATAATAGT TCCACATTGGAATTGGTACAAAGCCAATTTCAAGAGAAGTTAAGA GGAAAGAAGTACTTCCTTGTTCTTGATGATGTATGGAACGAAGAT CGTGAGAAGTGGCTTCCTTTGGAAGAGTTGTTAATGTTGGGTCAA

GGGGGAAGCAAGGTTGTAGTGACCGCACGTTCAGAGAAGACAGC AAATGTCATAGGGAAAAGACATTTTTATACACTGGAATGTTTGTC ACCAGATTATTCATGGAGCTTATTTGAAATGTCGGCTTTTCAGAAA GGGCATGAGCAGGAAAACCATCACGAACTAGTTGATATTGGGAAA AAGATTGTTGAAAAATGTTATAACAATCCACTTGCTATAACGGTG GTAGGAAGTCTTCTTTATGGAGAGGAGATAAGTAAGTGGCGGTCA TTTGAAATGAGTGAGTTGGCCAAAATTGGCAATGGGGATAATAAG ATTTTGCCGATATTAAAGCTCAGTTACCATAATCTTATACCCTCGT TGAAGAGTTGCTTCAGTTATTGTGCAGTGTTTCCCAAGGATCATGA AATAAAGAAGGAGATGTTGATTGATCTTTGGATAGCACAAGGATA CGTTGTGGCACTTGATGGAGGTCAAAGTATAGAAGATGCTGCCGA AGAACATTTTGTAATTTTGTTACGGAGATGTTTCTTTCAAGATGTA AAGAAGGATGAATATGGTGATGTTGATTCTGTTAAAATCCACGAC TTGATGCACGATGTCGCCCAAGAAGTGGGGAGGGAGGAAATATGT GTAGTGAATGATAATACAAAGAACTTGGGTGATAAAATCCGTCAT GTACATGGTGATGTCAATAGATATGCACAAAGAGTCTCTCTGTGT AGCCATAGCCATAAGATTCGTTCGTATATTGGTGGTGATTGTGAAA AACGTTGTGTGGATACACTAATAGACAAGTGGATGTGTCTTAGGA TGTTGGACTTGTCATGGTCGGATGTTAAAAATTTGCCTAATTCAAT AGGTAAATTGTTGCACTTGAGGTATCTTAACCTGTCAGATAATAGA AATCTAAAGATACTTCCTGATGCAATTACAAGACTGCATAATTTGC AGACACTGCTTTTAGAAGATTGCAGAAGTTTAAAGGAGTTGCCAA AAGATTTTTGCAAATTGGTCAAACTGAGGCACTTGGAATTACAGG GTTGTCATGATTTGATTGGTATGCCATTTGGAATGGATAAGCTAAC TAGTCTTAGAATACTACCAAACATTGTGGTGGGTAGGAAGGAACA AAGTGATGATGAGCTGAAAGCCCTAAAAGGCCTCACCGAGATAAA AGGCTCCATTTCTATCAGAATCTATTCAAAGTATAGAATAGTTGAA GGCATGAATGACACAGGAGGAGCTGCTTATTTGAAGAGCATGAAA CATCTCAGGGAGATTGATATTACATTTTTGGGTGAATGTGTTGGCC CTGAAGCTGTATTGGAAACCTTAGAGCCACCTTCAAATATCAAGA GCTTATATATATATAATTACAGTGGTACAACAATTCCAGTATGGGG AAGAGCAGAGATTAATTGGGCAATCTCCCTCTCACATCTCGTCGA CATCCAGCTTAGTTGTTGTAGTAATTTGCAGGAGATGCCAGTGCTG AGTAAACTGCCTCATTTGAAATCGCTGAAACTTGGATGGTTGGAT AACTTAGAGTACATGGAGAGTAGCAGTAGCAGTGACACAGAAGC AGCAACACCAGAATTACCAACATTCTTCCCTTCCCTTGAAAAACTT ACTTTACAGCATCTGGAAAAGTTGAAGGGTTTTGGGAACAGGAGA TCGAGTAGTTTTCCCCGCCTCTCTGAATTGGAAATCAAGAAATGCC CAGATCTAACGTCATTTCCTTCTTGTCCAAGCCTTGAGAAGTTGGA ATTGAAAGAAAGCAATGAAGCATTGCAAATAATAGTAAAAATAA CAACAAGAGGTAAAGAAAAAGAAGAGAACAATAATGCTGGTGTT AGAAATTCACAAGATGATGACAAAGTCAAATTACGGAAGATGGTG ATAGACAATCTGGGTTATCTCACGGGGGTTGATATTAGATTTGATG ATAGAGAAGGTGGATTTGTTAACCCTGAAGCTGTGTTGGCAACCC TAGAGCCACCTTCAAATATCAAGAGCTTATCTATACATCGTTTTGA TGGTAAAACACTTCCAGTATGGGGAAGAGCAGAGATTAATTGGGC AATCTCCCTCTCACATCTTGTCGACATCCAGCTTTGGCATTGTCGT AATTTGCAGGAGATGCCAGTGCTGAGTAAACTGCCTCATTTGAAA TCACTGGAACTTTATAATTTGATTAGTTTAGAGTACATGGAGAGCA CAAGCAGAAGCAGTAGCAGTGACACAGAAGCAGCAACACCAGAA TTACCAACATTCTTCCCTTCCCTTGAAAAACTTAGACTTTGGTATCT GGAAAAGTTGAAGGGTTTGGGGAACAGGAGACCGAGTAGTTTTCC CCGCCTCTCTGAATTGGAAATCTGGGAATGCCCAGATCTAACGTG GTTTCCTCCTTGTCCAAGCCTTAAAACGTTGAAATTGGAAAAAAAC AATGAAGCGTTGCAAATAATAGTAAAAATAACAACAACAAGAGG TAAAGAAGAAAAAGAAGAAGACAAGAATGCTGGTGTTGGAAATT CACAAGATGATGACAATGTCAAATTACGGAAGGTGGAAATAGAC AATGTGAGTTATCTCAAATCACTGCCCACAAATTGTCTTACTCACC TCAAAATAACTGGAATAGATTACAGGGAGGGGGAGATTGAATCA GATTCCGTGGAGGAGGAGATTGAATTGGAAGTTGGGGAGGCATTT CAGAAGTGTGCATCTTCTTTGAGAAGCCTCATCATAATCGGAAATC ACGGAATAAATAAAGTGATGAGACTGTCTGGAAGAACAGGGTTG GAGCATTTCACTCTGTTGGACTCACTCAAATTTTCAAAGATAGAAG ACCAGGAAGATGAGGGCGAAGACAACATCATATTCTGGAAATCCT TTCCTCAAAACCTTCGCAGTTTGAGAATTAAAGACTCTGACAAAAT GACAAGTTTGCCCATGGGGATGCAGTACTTAACCTCCCTCCAAAC CCTCGAACTATCATATTGTGATGAATTGAATTCCCTTCCAGAATGG ATAAGCAGCTTATCATCTCTTCAATACCTGCGCATATACTACTGTC CAGCCCTGAAATCACTACCAGAAGCAATGCGGAACCTCACCTCCC TTCAGACACTTGGGATATCGGATTGTCCAGACCTAGTTAAAAGAT GCAGAAAACCCAACGGCAAGGACTATCCCAAAATTCAACACATCC CCAAAATTGTAAGTCATTGCAGAAAGTAATTTATTCATTTATATTT ATTTTATGCTTAGAATGATATACGCAGTCGTCCTTTGGTTTCAAAT CTTGAATTTGGTTTTTGTTTTCTTTCTTTGTTTCTTTATTCAACACCA GCCCATTTATGATTGATTCATTAAAAAAAGGATGGAGTTTTATGGA TTTGAAGAAGACAACGAATTGAGATTCCTGGGGTTTTCTTTTTGTT GGGGTTGGATTTCATGTATATGTTGCTGATTAAATACGAGACTGAT GATGATGATGTGTTTATGGGTTTTAAATCAGATTAAATATATGGGA AATGCAAGTTAATTTGGGATGCACATAAGGTGTTTGCTGAAATGT CTATGAGAAATGTTGTTTCTTGGACTTAGAATGATATACACTGTCG TCCTTTGGTTTCCAATCTTACATTTGGTTTGTGTTTTCTTAGTTTGTT TCTTTAATCAACACCAACCCGTTTTTTTTAAACTACCTGCAACTAC TAATTTACGTTTACCCTGTATCTCAGGTACTAAATGAATATTGGTG ATTTTCAGTTACTCAACACTAGCTTGATCCTGAACGCACCCAACCT TCAGGTTAGAATCCGGCTTACTCATCCTTTTGTCCAGTTTTCAAGT AATTGTTTTGGCAGGATCAATTCTCTAATTGTTGTACACCGTATAT TGCAATTTATAGTGACTACAGTTAATGAATGTTTACAAAAAATTAG TCATGTAAAAACTTCTTCTCTGTCCATTACATAAACTCTTTTTCTCT TTCTAACTTATCATGTTCATGTCTAAACAATTAAACATGCTCACAT CAATGTTCATTTAAGCTAACTTACTTCTGTAAGAGAGCGAGCTAGT TAAAAACTCCTTTAACTTTCTGTTTTATACTCAGGACATGGATTGA TGCAAGCATGAAGAACTTCGGGAATTTGCTAAAACTCTACCAAAG CGATGAGAGTTTGGACTTTATTTCACTTGAAGTCAGGGACTGTCAA CAAAGCCACAGTGTGCATGTTGGCTGTTTCACTTGGACGATAAAA AGGTTTATTTAATTGTTTTCCTAAGTGTATTTGGCTTACAAGCTTTT ACTTTTCACTTGAAAGGGTTTTTCTTGTTTTAAGCTTTTCGAATTAG AGTTTTCGGTTGAAGTAAGAGTAGTCGTATTAGTCTTTTACCTAAG GAAGACTCTTTTTTGTAATTTTCAGACTATGCAATTCAAGTTTTCG AGTGTTTTCTTGCTTGTGTGATTGTGAGTTGGTGAATTCGTCTTTCA TACATTTTGAGATTATCAGAAGCTTTATGCTCCACCGGTAGTCTAG TACCTTTTCTGTTACTGTGCAGGGAAGTAATCTGGTACCTTCTATA TATATGGAAAAACATACATTATACATTATGCAAAATTCTTACAGGT TAGTTACTTCCTGGAACTTCATTTACACTTAGTTTTTTTTGTTCCAT TCCCTCGGAATCAAGTCATTCCCTCTGAGAAATATGTAATGAACTT CTGTATGTTGCTGTTTGGTTCCTGTTTTAATCTTCAATTTTCTTGTA TAGTTACAGCTGCATTTACAATGAAGTTTAAGCAGACACTCTCTTT ATATAGTGCCTCTTTCTGGAGCACCGTAGAGCTGTCTGTGGTTGAT CACCATCTGCTGCCGAGAGATTCAGCAATCGCGTGTTTGATCAGGT

AAAAGTTTTTATGTCAATGTGTTTTTTTTTCCGTTTGATCAATTTAT GTCTGTATTCAGATTCTTATCTTCTTACAGTAGCATAACACATTGTT TCTTTCATTTATGTAAACTGTTTCAAGATTACAGAGATGTATGCTT CAGTCGACATTGATGATAACTTAAGATGGCATTCCTACAACAGTT GCAGGCGCATTCTAACTCCGGCAATTCTAGTTAGGCAAGAGGAGC ATTGCCAATACCTGCCACCTCTGGGATTTACTATACCAGGGTTGAA GTTTATGGAAGACACCAGCTATGCACAAGCCTTCAAGGGGTCATC CTACATAACAAGTTGAACCAACCAATTGCTTGTTGGTTCAGTGGTA ATTGAAGCTGAATTTGGTAGGGATGGCCCGTGTTCGATCCCCACA ACAACAATTGGGAGGGGACTGGAACCTATCCACACAGAACTCGCC CTGAATCCGGATTAGCCCTAAGGGTGAACGGGGTGCTAACACCAA AAAAAAAAACATAACAAGTTGAACCAAACATACTTTGTTTGAATT GAAGATTTAGTGATTTCATTTGATCGATTGAGATGTCTTATTATAA GCGTATATGCTCTTGGATTTGGCCACTTAGGTGTTGTTTGACAATT GGACATTAACTCGCTTTTATATTTTCTTTTCTCTTAGGAAAGGTGAT CCTGAGAATTTATATTGGAACACTTTTTTTTTCTCACTAGCTTTAAA AAAGTGTTCTGTGTTACCTGCAATTCAATTTGATTATTTTTCACATA GTTTTACCTGAAAAAGTGTTACCTGAAAAAGTGTTACCTGAAAAT CAACTGACATAAGTTTTTGTTTGGATCCAATTAAGGACACTAGATA AATCGGAATAAATAATCAACCAATTAAGTACTTCATAATTAAATA TGAAGTGTATTATTATCTTATGCTTGTGACATTGAAGGATGTTATG ATATTTTAACTCAATACCTTGCAAAATATACTGGTTAAATTTCTTA ACAAGGTAACTTGGCAACA SEQ ID No:2: ATGGCCGAAATCGGATACTCGGTTTGTGCGAAACTCATCGAAGTG ATTGGCAGTGAGCTGATCAAAGAGATTTGTGACACATGGGGTTAC cdsalpha-WOLF AAATCTCTTCTTGAGGACCTCAACAAAACTGTATTGACGGTCAGG AACGTTCTCATTCAAGCCGGGGTGATGCGGGAGCTTACTAGTGAA CAACAAGGTTTCATTGCAGACCTTAAAGATGTTGTTTATGATGCTG ATGACTTGTTCGACAAGTTACTCACTCGTGCTGAGCGAAAACAGA TTGATGGAAACGAAATCTCTGAAAAGGTACGTCGTTTCTTTTCCTC TAGTAACAAGATCGGTCAAGCTTACTACATGTCTCGTAAGGTTAA GGAAATTAAGAAGCAGTTGGATGAAATTGTTGATAGGCATACAAA ATTTGGGTTTAGTGCCGAGTTTATACCTGTTTGTAGGGAAAGGGGG AACGAGAGGGAAACACGTTCATATATAGATGTCAAGAATATTCTT GGGAGGGATAAAGATAAGAATGATATCATAGATAGGTTGCTTAAT CGTAATGGTAATGAAGCTTGTAGTTTCCTGACCATAGTGGGAGCG GGAGGATTGGGAAAAACTGCTCTTGCACAACTTGTGTTCAATGAT GAAAGGGTCAAAATTGAGTTCCATGATTTGAGGTATTGGGTTTGT GTCTCTGATCAAGATGGGGGCCAATTTGATGTGAAAGAAATCCTT TGTAAGATTTTAGAGGTGGTTACTAAGGAGAAAGTTGATAATAGT TCCACATTGGAATTGGTACAAAGCCAATTTCAAGAGAAGTTAAGA GGAAAGAAGTACTTCCTTGTTCTTGATGATGTATGGAACGAAGAT CGTGAGAAGTGGCTTCCTTTGGAAGAGTTGTTAATGTTGGGTCAA GGGGGAAGCAAGGTTGTAGTGACCGCACGTTCAGAGAAGACAGC AAATGTCATAGGGAAAAGACATTTTTATACACTGGAATGTTTGTC ACCAGATTATTCATGGAGCTTATTTGAAATGTCGGCTTTTCAGAAA GGGCATGAGCAGGAAAACCATCACGAACTAGTTGATATTGGGAAA AAGATTGTTGAAAAATGTTATAACAATCCACTTGCTATAACGGTG GTAGGAAGTCTTCTTTATGGAGAGGAGATAAGTAAGTGGCGGTCA TTTGAAATGAGTGAGTTGGCCAAAATTGGCAATGGGGATAATAAG ATTTTGCCGATATTAAAGCTCAGTTACCATAATCTTATACCCTCGT TGAAGAGTTGCTTCAGTTATTGTGCAGTGTTTCCCAAGGATCATGA AATAAAGAAGGAGATGTTGATTGATCTTTGGATAGCACAAGGATA CGTTGTGGCACTTGATGGAGGTCAAAGTATAGAAGATGCTGCCGA

AGAACATTTTGTAATTTTGTTACGGAGATGTTTCTTTCAAGATGTA AAGAAGGATGAATATGGTGATGTTGATTCTGTTAAAATCCACGAC TTGATGCACGATGTCGCCCAAGAAGTGGGGAGGGAGGAAATATGT GTAGTGAATGATAATACAAAGAACTTGGGTGATAAAATCCGTCAT GTACATGGTGATGTCAATAGATATGCACAAAGAGTCTCTCTGTGT AGCCATAGCCATAAGATTCGTTCGTATATTGGTGGTGATTGTGAAA AACGTTGTGTGGATACACTAATAGACAAGTGGATGTGTCTTAGGA TGTTGGACTTGTCATGGTCGGATGTTAAAAATTTGCCTAATTCAAT AGGTAAATTGTTGCACTTGAGGTATCTTAACCTGTCAGATAATAGA AATCTAAAGATACTTCCTGATGCAATTACAAGACTGCATAATTTGC AGACACTGCTTTTAGAAGATTGCAGAAGTTTAAAGGAGTTGCCAA AAGATTTTTGCAAATTGGTCAAACTGAGGCACTTGGAATTACAGG GTTGTCATGATTTGATTGGTATGCCATTTGGAATGGATAAGCTAAC TAGTCTTAGAATACTACCAAACATTGTGGTGGGTAGGAAGGAACA AAGTGATGATGAGCTGAAAGCCCTAAAAGGCCTCACCGAGATAAA AGGCTCCATTTCTATCAGAATCTATTCAAAGTATAGAATAGTTGAA GGCATGAATGACACAGGAGGAGCTGCTTATTTGAAGAGCATGAAA CATCTCAGGGAGATTGATATTACATTTTTGGGTGAATGTGTTGGCC CTGAAGCTGTATTGGAAACCTTAGAGCCACCTTCAAATATCAAGA GCTTATATATATATAATTACAGTGGTACAACAATTCCAGTATGGGG AAGAGCAGAGATTAATTGGGCAATCTCCCTCTCACATCTCGTCGA CATCCAGCTTAGTTGTTGTAGTAATTTGCAGGAGATGCCAGTGCTG AGTAAACTGCCTCATTTGAAATCGCTGAAACTTGGATGGTTGGAT AACTTAGAGTACATGGAGAGTAGCAGTAGCAGTGACACAGAAGC AGCAACACCAGAATTACCAACATTCTTCCCTTCCCTTGAAAAACTT ACTTTACAGCATCTGGAAAAGTTGAAGGGTTTTGGGAACAGGAGA TCGAGTAGTTTTCCCCGCCTCTCTGAATTGGAAATCAAGAAATGCC CAGATCTAACGTCATTTCCTTCTTGTCCAAGCCTTGAGAAGTTGGA ATTGAAAGAAAGCAATGAAGCATTGCAAATAATAGTAAAAATAA CAACAAGAGGTAAAGAAAAAGAAGAGAACAATAATGCTGGTGTT AGAAATTCACAAGATGATGACAAAGTCAAATTACGGAAGATGGTG ATAGACAATCTGGGTTATCTCACGGGGGTTGATATTAGATTTGATG ATAGAGAAGGTGGATTTGTTAACCCTGAAGCTGTGTTGGCAACCC TAGAGCCACCTTCAAATATCAAGAGCTTATCTATACATCGTTTTGA TGGTAAAACACTTCCAGTATGGGGAAGAGCAGAGATTAATTGGGC AATCTCCCTCTCACATCTTGTCGACATCCAGCTTTGGCATTGTCGT AATTTGCAGGAGATGCCAGTGCTGAGTAAACTGCCTCATTTGAAA TCACTGGAACTTTATAATTTGATTAGTTTAGAGTACATGGAGAGCA CAAGCAGAAGCAGTAGCAGTGACACAGAAGCAGCAACACCAGAA TTACCAACATTCTTCCCTTCCCTTGAAAAACTTAGACTTTGGTATCT GGAAAAGTTGAAGGGTTTGGGGAACAGGAGACCGAGTAGTTTTCC CCGCCTCTCTGAATTGGAAATCTGGGAATGCCCAGATCTAACGTG GTTTCCTCCTTGTCCAAGCCTTAAAACGTTGAAATTGGAAAAAAAC AATGAAGCGTTGCAAATAATAGTAAAAATAACAACAACAAGAGG TAAAGAAGAAAAAGAAGAAGACAAGAATGCTGGTGTTGGAAATT CACAAGATGATGACAATGTCAAATTACGGAAGGTGGAAATAGAC AATGTGAGTTATCTCAAATCACTGCCCACAAATTGTCTTACTCACC TCAAAATAACTGGAATAGATTACAGGGAGGGGGAGATTGAATCA GATTCCGTGGAGGAGGAGATTGAATTGGAAGTTGGGGAGGCATTT CAGAAGTGTGCATCTTCTTTGAGAAGCCTCATCATAATCGGAAATC ACGGAATAAATAAAGTGATGAGACTGTCTGGAAGAACAGGGTTG GAGCATTTCACTCTGTTGGACTCACTCAAATTTTCAAAGATAGAAG ACCAGGAAGATGAGGGCGAAGACAACATCATATTCTGGAAATCCT TTCCTCAAAACCTTCGCAGTTTGAGAATTAAAGACTCTGACAAAAT

GACAAGTTTGCCCATGGGGATGCAGTACTTAACCTCCCTCCAAAC CCTCGAACTATCATATTGTGATGAATTGAATTCCCTTCCAGAATGG ATAAGCAGCTTATCATCTCTTCAATACCTGCGCATATACTACTGTC CAGCCCTGAAATCACTACCAGAAGCAATGCGGAACCTCACCTCCC TTCAGACACTTGGGATATCGGATTGTCCAGACCTAGTTAAAAGAT GCAGAAAACCCAACGGCAAGGACTATCCCAAAATTCAACACATCC CCAAAATTGTACTAAATGAATATTGGTGA SEQ ID No:3: ATGGCCGAAATCGGATACTCGGTTTGTGCGAAACTCATCGAAGTG ATTGGCAGTGAGCTGATCAAAGAGATTTGTGACACATGGGGTTAC cdsofalpha- AAATCTCTTCTTGAGGACCTCAACAAAACTGTATTGACGGTCAGG WOLF 15 AACGTTCTCATTCAAGCCGGGGTGATGCGGGAGCTTACTAGTGAA CAACAAGGTTTCATTGCAGACCTTAAAGATGTTGTTTATGATGCTG (isoform 1) ATGACTTGTTCGACAAGTTACTCACTCGTGCTGAGCGAAAACAGA TTGATGGAAACGAAATCTCTGAAAAGGTACGTCGTTTCTTTTCCTC TAGTAACAAGATCGGTCAAGCTTACTACATGTCTCGTAAGGTTAA GGAAATTAAGAAGCAGTTGGATGAAATTGTTGATAGGCATACAAA ATTTGGGTTTAGTGCCGAGTTTATACCTGTTTGTAGGGAAAGGGGG AACGAGAGGGAAACACGTTCATATATAGATGTCAAGAATATTCTT GGGAGGGATAAAGATAAGAATGATATCATAGATAGGTTGCTTAAT CGTAATGGTAATGAAGCTTGTAGTTTCCTGACCATAGTGGGAGCG GGAGGATTGGGAAAAACTGCTCTTGCACAACTTGTGTTCAATGAT GAAAGGGTCAAAATTGAGTTCCATGATTTGAGGTATTGGGTTTGT GTCTCTGATCAAGATGGGGGCCAATTTGATGTGAAAGAAATCCTT TGTAAGATTTTAGAGGTGGTTACTAAGGAGAAAGTTGATAATAGT TCCACATTGGAATTGGTACAAAGCCAATTTCAAGAGAAGTTAAGA GGAAAGAAGTACTTCCTTGTTCTTGATGATGTATGGAACGAAGAT CGTGAGAAGTGGCTTCCTTTGGAAGAGTTGTTAATGTTGGGTCAA GGGGGAAGCAAGGTTGTAGTGACCGCACGTTCAGAGAAGACAGC AAATGTCATAGGGAAAAGACATTTTTATACACTGGAATGTTTGTC ACCAGATTATTCATGGAGCTTATTTGAAATGTCGGCTTTTCAGAAA GGGCATGAGCAGGAAAACCATCACGAACTAGTTGATATTGGGAAA AAGATTGTTGAAAAATGTTATAACAATCCACTTGCTATAACGGTG GTAGGAAGTCTTCTTTATGGAGAGGAGATAAGTAAGTGGCGGTCA TTTGAAATGAGTGAGTTGGCCAAAATTGGCAATGGGGATAATAAG ATTTTGCCGATATTAAAGCTCAGTTACCATAATCTTATACCCTCGT TGAAGAGTTGCTTCAGTTATTGTGCAGTGTTTCCCAAGGATCATGA AATAAAGAAGGAGATGTTGATTGATCTTTGGATAGCACAAGGATA CGTTGTGGCACTTGATGGAGGTCAAAGTATAGAAGATGCTGCCGA AGAACATTTTGTAATTTTGTTACGGAGATGTTTCTTTCAAGATGTA AAGAAGGATGAATATGGTGATGTTGATTCTGTTAAAATCCACGAC TTGATGCACGATGTCGCCCAAGAAGTGGGGAGGGAGGAAATATGT GTAGTGAATGATAATACAAAGAACTTGGGTGATAAAATCCGTCAT GTACATGGTGATGTCAATAGATATGCACAAAGAGTCTCTCTGTGT AGCCATAGCCATAAGATTCGTTCGTATATTGGTGGTGATTGTGAAA AACGTTGTGTGGATACACTAATAGACAAGTGGATGTGTCTTAGGA TGTTGGACTTGTCATGGTCGGATGTTAAAAATTTGCCTAATTCAAT AGGTAAATTGTTGCACTTGAGGTATCTTAACCTGTCAGATAATAGA AATCTAAAGATACTTCCTGATGCAATTACAAGACTGCATAATTTGC AGACACTGCTTTTAGAAGATTGCAGAAGTTTAAAGGAGTTGCCAA AAGATTTTTGCAAATTGGTCAAACTGAGGCACTTGGAATTACAGG GTTGTCATGATTTGATTGGTATGCCATTTGGAATGGATAAGCTAAC TAGTCTTAGAATACTACCAAACATTGTGGTGGGTAGGAAGGAACA AAGTGATGATGAGCTGAAAGCCCTAAAAGGCCTCACCGAGATAAA AGGCTCCATTTCTATCAGAATCTATTCAAAGTATAGAATAGTTGAA

GGCATGAATGACACAGGAGGAGCTGCTTATTTGAAGAGCATGAAA CATCTCAGGGAGATTGATATTACATTTTTGGGTGAATGTGTTGGCC CTGAAGCTGTATTGGAAACCTTAGAGCCACCTTCAAATATCAAGA GCTTATATATATATAATTACAGTGGTACAACAATTCCAGTATGGGG AAGAGCAGAGATTAATTGGGCAATCTCCCTCTCACATCTCGTCGA CATCCAGCTTAGTTGTTGTAGTAATTTGCAGGAGATGCCAGTGCTG AGTAAACTGCCTCATTTGAAATCGCTGAAACTTGGATGGTTGGAT AACTTAGAGTACATGGAGAGTAGCAGTAGCAGTGACACAGAAGC AGCAACACCAGAATTACCAACATTCTTCCCTTCCCTTGAAAAACTT ACTTTACAGCATCTGGAAAAGTTGAAGGGTTTTGGGAACAGGAGA TCGAGTAGTTTTCCCCGCCTCTCTGAATTGGAAATCAAGAAATGCC CAGATCTAACGTCATTTCCTTCTTGTCCAAGCCTTGAGAAGTTGGA ATTGAAAGAAAGCAATGAAGCATTGCAAATAATAGTAAAAATAA CAACAAGAGGTAAAGAAAAAGAAGAGAACAATAATGCTGGTGTT AGAAATTCACAAGATGATGACAAAGTCAAATTACGGAAGATGGTG ATAGACAATCTGGGTTATCTCACGGGGGTTGATATTAGATTTGATG ATAGAGAAGGTGGATTTGTTAACCCTGAAGCTGTGTTGGCAACCC TAGAGCCACCTTCAAATATCAAGAGCTTATCTATACATCGTTTTGA TGGTAAAACACTTCCAGTATGGGGAAGAGCAGAGATTAATTGGGC AATCTCCCTCTCACATCTTGTCGACATCCAGCTTTGGCATTGTCGT AATTTGCAGGAGATGCCAGTGCTGAGTAAACTGCCTCATTTGAAA TCACTGGAACTTTATAATTTGATTAGTTTAGAGTACATGGAGAGCA CAAGCAGAAGCAGTAGCAGTGACACAGAAGCAGCAACACCAGAA TTACCAACATTCTTCCCTTCCCTTGAAAAACTTAGACTTTGGTATCT GGAAAAGTTGAAGGGTTTGGGGAACAGGAGACCGAGTAGTTTTCC CCGCCTCTCTGAATTGGAAATCTGGGAATGCCCAGATCTAACGTG GTTTCCTCCTTGTCCAAGCCTTAAAACGTTGAAATTGGAAAAAAAC AATGAAGCGTTGCAAATAATAGTAAAAATAACAACAACAAGAGG TAAAGAAGAAAAAGAAGAAGACAAGAATGCTGGTGTTGGAAATT CACAAGATGATGACAATGTCAAATTACGGAAGGTGGAAATAGAC AATGTGAGTTATCTCAAATCACTGCCCACAAATTGTCTTACTCACC TCAAAATAACTGGAATAGATTACAGGGAGGGGGAGATTGAATCA GATTCCGTGGAGGAGGAGATTGAATTGGAAGTTGGGGAGGCATTT CAGAAGTGTGCATCTTCTTTGAGAAGCCTCATCATAATCGGAAATC ACGGAATAAATAAAGTGATGAGACTGTCTGGAAGAACAGGGTTG GAGCATTTCACTCTGTTGGACTCACTCAAATTTTCAAAGATAGAAG ACCAGGAAGATGAGGGCGAAGACAACATCATATTCTGGAAATCCT TTCCTCAAAACCTTCGCAGTTTGAGAATTAAAGACTCTGACAAAAT GACAAGTTTGCCCATGGGGATGCAGTACTTAACCTCCCTCCAAAC CCTCGAACTATCATATTGTGATGAATTGAATTCCCTTCCAGAATGG ATAAGCAGCTTATCATCTCTTCAATACCTGCGCATATACTACTGTC CAGCCCTGAAATCACTACCAGAAGCAATGCGGAACCTCACCTCCC TTCAGACACTTGGGATATCGGATTGTCCAGACCTAGTTAAAAGAT GCAGAAAACCCAACGGCAAGGACTATCCCAAAATTCAACACATCC CCAAAATTTTACTCAACACTAGCTTGATCCTGAACGCACCCAACCT TCAGGACATGGATTGA SEQ ID No:4: MAEIGYSVCAKLIEVIGSELIKEICDTWGYKSLLEDLNKTVLTVRNVLI QAGVMRELTSEQQGFIADLKDVVYDADDLFDKLLTRAERKQIDGNEI proteinsequence SEKVRRFFSSSNKIGQAYYMSRKVKEIKKQLDEIVDRHTKFGFSAEFIP of alpha-WOLF VCRERGNERETRSYIDVKNILGRDKDKNDIIDRLLNRNGNEACSFLTI VGAGGLGKTALAQLVFNDERVKIEFHDLRYWVCVSDQDGGQFDVK EILCKILEVVTKEKVDNSSTLELVQSQFQEKLRGKKYFLVLDDVWNE DREKWLPLEELLMLGQGGSKVVVTARSEKTANVIGKRHFYTLECLSP DYSWSLFEMSAFQKGHEQENHHELVDIGKKIVEKCYNNPLAITVVGS

LLYGEEISKWRSFEMSELAKIGNGDNKILPILKLSYHNLIPSLKSCFSYC AVFPKDHEIKKEMLIDLWIAQGYVVALDGGQSIEDAAEEHFVILLRRC FFQDVKKDEYGDVDSVKIHDLMHDVAQEVGREEICVVNDNTKNLGD KIRHVHGDVNRYAQRVSLCSHSHKIRSYIGGDCEKRCVDTLIDKWMC LRMLDLSWSDVKNLPNSIGKLLHLRYLNLSDNRNLKILPDAITRLHNL QTLLLEDCRSLKELPKDFCKLVKLRHLELQGCHDLIGMPFGMDKLTS LRILPNIVVGRKEQSDDELKALKGLTEIKGSISIRIYSKYRIVEGMNDT GGAAYLKSMKHLREIDITFLGECVGPEAVLETLEPPSNIKSLYIYNYSG TTIPVWGRAEINWAISLSHLVDIQLSCCSNLQEMPVLSKLPHLKSLKL GWLDNLEYMESSSSSDTEAATPELPTFFPSLEKLTLQHLEKLKGFGNR RSSSFPRLSELEIKKCPDLTSFPSCPSLEKLELKESNEALQIIVKITTRGK EKEENNNAGVRNSQDDDKVKLRKMVIDNLGYLTGVDIRFDDREGGF VNPEAVLATLEPPSNIKSLSIHRFDGKTLPVWGRAEINWAISLSHLVDI QLWHCRNLQEMPVLSKLPHLKSLELYNLISLEYMESTSRSSSSDTEAA TPELPTFFPSLEKLRLWYLEKLKGLGNRRPSSFPRLSELEIWECPDLTW FPPCPSLKTLKLEKNNEALQIIVKITTTRGKEEKEEDKNAGVGNSQDD DNVKLRKVEIDNVSYLKSLPTNCLTHLKITGIDYREGEIESDSVEEEIE LEVGEAFQKCASSLRSLIIIGNHGINKVMRLSGRTGLEHFTLLDSLKFS KIEDQEDEGEDNIIFWKSFPQNLRSLRIKDSDKMTSLPMGMQYLTSLQ TLELSYCDELNSLPEWISSLSSLQYLRIYYCPALKSLPEAMRNLTSLQT LGISDCPDLVKRCRKPNGKDYPKIQHIPKIVLNEYW*

SEQ ID No:5: MAEIGYSVCAKLIEVIGSELIKEICDTWGYKSLLEDLNKTVLTVRNVLI QAGVMRELTSEQQGFIADLKDVVYDADDLFDKLLTRAERKQIDGNEI proteinsequence SEKVRRFFSSSNKIGQAYYMSRKVKEIKKQLDEIVDRHTKFGFSAEFIP of alpha-WOLF VCRERGNERETRSYIDVKNILGRDKDKNDIIDRLLNRNGNEACSFLTI 15 (isoform 1) VGAGGLGKTALAQLVFNDERVKIEFHDLRYWVCVSDQDGGQFDVK EILCKILEVVTKEKVDNSSTLELVQSQFQEKLRGKKYFLVLDDVWNE DREKWLPLEELLMLGQGGSKVVVTARSEKTANVIGKRHFYTLECLSP DYSWSLFEMSAFQKGHEQENHHELVDIGKKIVEKCYNNPLAITVVGS LLYGEEISKWRSFEMSELAKIGNGDNKILPILKLSYHNLIPSLKSCFSYC AVFPKDHEIKKEMLIDLWIAQGYVVALDGGQSIEDAAEEHFVILLRRC FFQDVKKDEYGDVDSVKIHDLMHDVAQEVGREEICVVNDNTKNLGD KIRHVHGDVNRYAQRVSLCSHSHKIRSYIGGDCEKRCVDTLIDKWMC LRMLDLSWSDVKNLPNSIGKLLHLRYLNLSDNRNLKILPDAITRLHNL QTLLLEDCRSLKELPKDFCKLVKLRHLELQGCHDLIGMPFGMDKLTS LRILPNIVVGRKEQSDDELKALKGLTEIKGSISIRIYSKYRIVEGMNDT GGAAYLKSMKHLREIDITFLGECVGPEAVLETLEPPSNIKSLYIYNYSG TTIPVWGRAEINWAISLSHLVDIQLSCCSNLQEMPVLSKLPHLKSLKL GWLDNLEYMESSSSSDTEAATPELPTFFPSLEKLTLQHLEKLKGFGNR RSSSFPRLSELEIKKCPDLTSFPSCPSLEKLELKESNEALQIIVKITTRGK EKEENNNAGVRNSQDDDKVKLRKMVIDNLGYLTGVDIRFDDREGGF VNPEAVLATLEPPSNIKSLSIHRFDGKTLPVWGRAEINWAISLSHLVDI QLWHCRNLQEMPVLSKLPHLKSLELYNLISLEYMESTSRSSSSDTEAA TPELPTFFPSLEKLRLWYLEKLKGLGNRRPSSFPRLSELEIWECPDLTW FPPCPSLKTLKLEKNNEALQIIVKITTTRGKEEKEEDKNAGVGNSQDD DNVKLRKVEIDNVSYLKSLPTNCLTHLKITGIDYREGEIESDSVEEEIE LEVGEAFQKCASSLRSLIIIGNHGINKVMRLSGRTGLEHFTLLDSLKFS KIEDQEDEGEDNIIFWKSFPQNLRSLRIKDSDKMTSLPMGMQYLTSLQ TLELSYCDELNSLPEWISSLSSLQYLRIYYCPALKSLPEAMRNLTSLQT LGISDCPDLVKRCRKPNGKDYPKIQHIPKILLNTSLILNAPNLQDMD* SEQ ID No:6: ACAAGTGGATGTGTCTTAGG

Forward primer

LRR domain (Alpha) SEQ ID No:7: TTCGCCCTCATCTTCCTGG Reverse primer

LRR domain SEQ ID No:8: TCACGTGGGTTGTGTTGT Forward primer

LRR domain (Beta)

SEQ ID No:9: ACAAGTGGATGTGTCTTAGGATGTTGGACTTGTCATGGTCGGATGT TAAAAATTTGCCTAATTCAATAGGTAAATTGTTGCACTTGAGGTAT Amplicon of CTTAACCTGTCAGATAATAGAAATCTAAAGATACTTCCTGATGCA LRR domain of ATTACAAGACTGCATAATTTGCAGACACTGCTTTTAGAAGATTGCA the alpha-WOLF GAAGTTTAAAGGAGTTGCCAAAAGATTTTTGCAAATTGGTCAAAC TGAGGCACTTGGAATTACAGGGTTGTCATGATTTGATTGGTATGCC 15 allele ATTTGGAATGGATAAGCTAACTAGTCTTAGAATACTACCAAACAT TGTGGTGGGTAGGAAGGAACAAAGTGATGATGAGCTGAAAGCCCT AAAAGGCCTCACCGAGATAAAAGGCTCCATTTCTATCAGAATCTA TTCAAAGTATAGAATAGTTGAAGGCATGAATGACACAGGAGGAGC TGCTTATTTGAAGAGCATGAAACATCTCAGGGAGATTGATATTAC ATTTTTGGGTGAATGTGTTGGCCCTGAAGCTGTATTGGAAACCTTA GAGCCACCTTCAAATATCAAGAGCTTATATATATATAATTACAGTG GTACAACAATTCCAGTATGGGGAAGAGCAGAGATTAATTGGGCAA TCTCCCTCTCACATCTCGTCGACATCCAGCTTAGTTGTTGTAGTAA TTTGCAGGAGATGCCAGTGCTGAGTAAACTGCCTCATTTGAAATC GCTGAAACTTGGATGGTTGGATAACTTAGAGTACATGGAGAGTAG CAGTAGCAGTGACACAGAAGCAGCAACACCAGAATTACCAACATT CTTCCCTTCCCTTGAAAAACTTACTTTACAGCATCTGGAAAAGTTG AAGGGTTTTGGGAACAGGAGATCGAGTAGTTTTCCCCGCCTCTCTG AATTGGAAATCAAGAAATGCCCAGATCTAACGTCATTTCCTTCTTG TCCAAGCCTTGAGAAGTTGGAATTGAAAGAAAGCAATGAAGCATT GCAAATAATAGTAAAAATAACAACAAGAGGTAAAGAAAAAGAAG AGAACAATAATGCTGGTGTTAGAAATTCACAAGATGATGACAAAG TCAAATTACGGAAGATGGTGATAGACAATCTGGGTTATCTCACGG GGGTTGATATTAGATTTGATGATAGAGAAGGTGGATTTGTTAACC CTGAAGCTGTGTTGGCAACCCTAGAGCCACCTTCAAATATCAAGA GCTTATCTATACATCGTTTTGATGGTAAAACACTTCCAGTATGGGG AAGAGCAGAGATTAATTGGGCAATCTCCCTCTCACATCTTGTCGAC ATCCAGCTTTGGCATTGTCGTAATTTGCAGGAGATGCCAGTGCTGA GTAAACTGCCTCATTTGAAATCACTGGAACTTTATAATTTGATTAG TTTAGAGTACATGGAGAGCACAAGCAGAAGCAGTAGCAGTGACA CAGAAGCAGCAACACCAGAATTACCAACATTCTTCCCTTCCCTTGA AAAACTTAGACTTTGGTATCTGGAAAAGTTGAAGGGTTTGGGGAA CAGGAGACCGAGTAGTTTTCCCCGCCTCTCTGAATTGGAAATCTGG GAATGCCCAGATCTAACGTGGTTTCCTCCTTGTCCAAGCCTTAAAA CGTTGAAATTGGAAAAAAACAATGAAGCGTTGCAAATAATAGTAA AAATAACAACAACAAGAGGTAAAGAAGAAAAAGAAGAAGACAA

GAATGCTGGTGTTGGAAATTCACAAGATGATGACAATGTCAAATT ACGGAAGGTGGAAATAGACAATGTGAGTTATCTCAAATCACTGCC CACAAATTGTCTTACTCACCTCAAAATAACTGGAATAGATTACAG GGAGGGGGAGATTGAATCAGATTCCGTGGAGGAGGAGATTGAATT GGAAGTTGGGGAGGCATTTCAGAAGTGTGCATCTTCTTTGAGAAG CCTCATCATAATCGGAAATCACGGAATAAATAAAGTGATGAGACT GTCTGGAAGAACAGGGTTGGAGCATTTCACTCTGTTGGACTCACTC AAATTTTCAAAGATAGAAGACCAGGAAGATGAGGGCGAA SEQ ID No:10: KWMCLRMLDLSWSDVKNLPNSIGKLLHLRYLNLSDNRNLKILPDAIT RLHNLQTLLLEDCRSLKELPKDFCKLVKLRHLELQGCHDLIGMPFGM amino acid DKLTSLRILPNIVVGRKEQSDDELKALKGLTEIKGSISIRIYSKYRIVEG sequence MNDTGGAAYLKSMKHLREIDITFLGECVGPEAVLETLEPPSNIKSLYI encoded by YNYSGTTIPVWGRAEINWAISLSHLVDIQLSCCSNLQEMPVLSKLPHL KSLKLGWLDNLEYMESSSSSDTEAATPELPTFFPSLEKLTLQHLEKLK amplicon of LRR GFGNRRSSSFPRLSELEIKKCPDLTSFPSCPSLEKLELKESNEALQIIVKI domain of alpha- TTRGKEKEENNNAGVRNSQDDDKVKLRKMVIDNLGYLTGVDIRFDD REGGFVNPEAVLATLEPPSNIKSLSIHRFDGKTLPVWGRAEINWAISLS WOLF 15 HLVDIQLWHCRNLQEMPVLSKLPHLKSLELYNLISLEYMESTSRSSSS DTEAATPELPTFFPSLEKLRLWYLEKLKGLGNRRPSSFPRLSELEIWEC PDLTWFPPCPSLKTLKLEKNNEALQIIVKITTTRGKEEKEEDKNAGVG NSQDDDNVKLRKVEIDNVSYLKSLPTNCLTHLKITGIDYREGEIESDS VEEEIELEVGEAFQKCASSLRSLIIIGNHGINKVMRLSGRTGLEHFTLL DSLKFSKIEDQEDEGE SEQ ID No:11: TCACGTGGGTTGTGTTGTCGATAGAGATCCAGAAATAGTCTTTTTA TGTAGCAATAAGATTCGTTCGTATATTAGCGGTCGCTGCATAAAG Amplicon of AATCCGGTGGATTCACAAATAGACAACTGGATGTGCCTTAGGGTG LRR domain of TTGGACTTGTCAGATTCATGTGTTAAAGATTTGTCTGATTCAATAG the beta-WOLF 0 GTAAGCTGCTGCACTTAAGGTATCTTAACCTCTCTTCTAATATAAA GTTGGAGATAATCCCTGATGCAATTACAAGACTGCATAACTTGCA allele GACACTACTTTTAGAAGATTGCAGAAGTTTAAAGGAGTTGCCAAA AGATTTTTGCAAATTGGTCAAACTGAGGCACTTGGAATTACAGGG TTGTCATGATTTGATTGGTATGTCATTTGGAATGGATAAGCTAACT AGTCTTAGAATACTACCAAACATTGTGGTGGGTAGGAAGGAACAA AGTGTTGATGATGAGCTGAAAGCCCTAAAAGGCCTCACCGAGATA AAAGGCTCCATTGATATCACAATCTATTCAAAATATAGAAGAGTT GAAGGCATGAATGGCACAGGAGGAGGAGCTGGGTATTTGAAGAG CATGAAACATCTCACGGGGGTTAATATTACATTTGATGAAGGTGG ATGTGTTAACCCTGAAGCTGTGTATTTGAAGAGCATGAAACATCTC ACGAGGGTTATTATTATATTTGATTATAAAGGTGGATGTGTTAACC CTGAAGCTGTGTTGGCAACCCTAGAGCCACCTTCAAATATCAAGA GGTTAGAGATGTGGCATTACAGTGGTACAACAATTCCAGTATGGG GAAGAGCAGAGATTAATTGGGCAATCTCCCTCTCACATCTTGTCG ACATCACGCTTGAAGATTGTTACAATTTGCAGGAGATGCCAGTGC TGAGTAAACTGCCTCATTTGAAATCACTGGAACTTACAGAGTTGG ATAACTTAGAGTACATGGAGAGTAGAAGCAGCAGCAGTAGCAGT GACACAGAAGCAGCAACACCAGAATTACCAACATTCTTCCCTTCC CTTGAAAAACTTACACTTTGGCGTCTGGACAAGTTGAAGGGTTTTG GGAACAGGAGATCGAGTAGTTTTCCCCGCCTCTCTAAATTGGAAA TCTGGAAATGTCCAGATCTAACGTCATTTCCTTCTTGTCCAAGCCT TGAAGAGTTGGAATTGAAAGAAAACAATGAAGCGTTGCAAATAAT AGTAAAAATAACAACAACAAGAGGTAAAGAAGAAAAAGAAGAA GACAAGAATGCTGGTGTTGGAAATTCACAAGATGATGACAATGTC AAATTATGGAAGGTGGAAATAGACAATCTGGGTTATCTCAAATCA

CTGCCCACAAATTGTCTGACTCACCTCGACCTTACAATAAGTGATT CCAAGGAGGGGGAGGGTGAATGGGAAGTTGGGGATGCATTTCAG AAGTGTGTATCTTCTTTGAGAAGCCTCACCATAATCGGAAATCACG GAATAAATAAAGTGAAGAGACTGTCTGGAAGAACAGGGTTGGAG CATTTCACTCTGTTGGAATCACTCAAACTTTCAGATATAGAAGACC AGGAAGATGAGGGCGAA SEQ ID No:12: HVGCVVDRDPEIVFLCSNKIRSYISGRCIKNPVDSQIDNWMCLRVLDL SDSCVKDLSDSIGKLLHLRYLNLSSNIKLEIIPDAITRLHNLQTLLLEDC amino acid RSLKELPKDFCKLVKLRHLELQGCHDLIGMSFGMDKLTSLRILPNIVV sequence GRKEQSVDDELKALKGLTEIKGSIDITIYSKYRRVEGMNGTGGGAGY LKSMKHLTGVNITFDEGGCVNPEAVYLKSMKHLTRVIIIFDYKGGCV encoded by NPEAVLATLEPPSNIKRLEMWHYSGTTIPVWGRAEINWAISLSHLVDI amplicon of LRR TLEDCYNLQEMPVLSKLPHLKSLELTELDNLEYMESRSSSSSSDTEAA domain Beta TPELPTFFPSLEKLTLWRLDKLKGFGNRRSSSFPRLSKLEIWKCPDLTS FPSCPSLEELELKENNEALQIIVKITTTRGKEEKEEDKNAGVGNSQDD Wolf 0 (Viroflay) DNVKLWKVEIDNLGYLKSLPTNCLTHLDLTISDSKEGEGEWEVGDAF QKCVSSLRSLTIIGNHGINKVKRLSGRTGLEHFTLLESLKLSDIEDQED EGE

The present invention will be further clarified in the Examples that follow and that are

given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES EXAMPLE1

Testingfor resistance to Peronosporafarinosa f. sp. spinaciaejinspinach plants

The resistance to downy mildew infection was assayed as described by Irish et al.

(2008; Phytopathol. 98: 894-900), using a differential set. Spinach plants of the invention were sown along with spinach plants from different other genotypes (see Table 3) in trays containing

Scotts Redi-Earth medium, and fertilized twice a week after seedling emergence with Osmocote

Peter's (13-13-13) fertilizer (Scotts). Plants were inoculated with a sporangial suspension (2.5 x

10 5 /ml) of a pathogenic race of Peronosporafarinosaf. sp. spinaciae at the first true leaf stage. In

this manner, 16 officially recognized pathogenic races were tested.

The inoculated plants were placed in a dew chamber at 18°C with 100% relative

humidity for a 24 h period, and then moved to a growth chamber at 18°C with a 12 h photoperiod

for 6 days. After 6 days, the plants were returned to the dew chamber for 24 h to induce

sporulation, and they were scored for disease reaction.

Plants for this specific test were scored as resistant, intermediately resistant, or

susceptible based on symptoms of chlorosis and signs of pathogen sporulation on the cotyledons

and true leaves, as described by Irish et al. (2007; PlantDis. 91: 1392-1396). Plants exhibiting no evidence of chlorosis and sporulation were in this specific test considered as resistant. Resistant

plants were re-inoculated to assess whether plants initially scored as resistant had escaped

infection, or whether they were truly resistant. Plants that showed only symptoms of chlorosis, or sporulation occurring only on the tips of the cotyledons were scored as intermediately resistant.

Plants showing more than these symptoms of downy mildew infection were scored as being

susceptible.

Table 1 shows the resistance of a plant carrying the alpha-WOLF 15 allele to each

one of these pathogenic races. Table 3 shows the differential set of spinach downy mildew races

and the resistance of various spinach varieties (hybrids) to each one of these pathogenic races. A

susceptible reaction is scored as "+" (indicating a successful infection by the fungus, with

sporulation occurring on the entire cotyledon), and resistance is depicted as "-" (absence of

sporulation on the cotyledons). A weak resistance response is indicated as "(-)",which in practice

means a slightly reduced level of infection (with only symptoms of chlorosis, or sporulation only

occurring on the tips of the cotyledons in the differential seedling test).

Table 3

Pfs:1 - -+

Pfs:2 - -+

Pfs: 3 - -+

Pfs:4 - - - -+

Pfs: 5 - -+ -

Pfs: 6 + + - - - +

Pfs:7 -++N

Pfs:08 - - - - -

Pfs:9 + + - - - - - - -

Pfs: 10 + + -++-+ -

Pfs:l + + - + - - -

Pfs: 12 + + - + + + - + - - -

Pfs:15 Pfs:13 + + E+ -NM +- - -- -- +- - - - -

Pfs:14 + + + + + + - + + -

Pfs: 15 + + + - - - - - + + -

Pfs:16 + + - + +- + +

EXAMPLE2 Amplification of the LRR domain-encoding region

The isolated genomic DNA of a spinach plant comprising the alpha-WOLF 15 allele,

of which a representative sample of seed was deposited with the NCIMB under NCIMB

accession number 42466 was used in polymerase chain reactions (PCR), using

forward primer ACAAGTGGATGTGTCTTAGG (SEQ ID No:6) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID No:7). The primer pair amplifies the LRR domain encoding region of an alpha-WOLF gene, and has been designed for selectively amplifying part of

a WOLF gene, and not of other CC-NBS-LRR protein-encoding genes.

PCR conditions for amplifying the LRR domain-encoding region of an alpha- WOLF gene using primers having SEQ ID No:6 and SEQ ID No:7 were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):

- 3 minutes at 95°C (initial denaturing step)

- 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C,

30 seconds annealing at 60°C, and 30 seconds extension at 72°C

- 2 minutes at 72°C (final extension step)

The isolated genomic DNA of a spinach plant of variety Viroflay comprising the beta

WOLF 0 allele was used in polymerase chain reactions (PCR), using forward primer

TCACGTGGGTTGTGTTGT (SEQ ID No:8) and reverse primer TTCGCCCTCATCTTCCTGG (SEQ ID No:7). The primer pair amplifies the LRR domain-encoding region of a beta-WOLF gene, and has been designed for selectively amplifying part of a WOLF gene, and not of other CC-NBS

LRR protein-encoding genes.

PCR conditions for amplifying the LRR domain-encoding region of a beta- WOLF

gene using primers having SEQ ID No:7 and SEQ ID No:8 were as follows, using Platinum Taq enzyme (Thermo Fisher Scientific):

- 3 minutes at 95°C (initial denaturing step)

- 40 amplification cycles, each cycle consisting of: 30 seconds denaturation at 95°C,

50 seconds annealing at 58°C and 50 seconds extension at 72°C

- 2 minutes at 72°C (final extension step)

The PCR products were visualized on agarose gel (not shown), and DNA was purified

from the PCR reaction. Subsequently the sequence of the PCR products was determined using

methods well known in the art.

The sequence of the LRR domain of the alpha WOLF 15 allele amplified by primers having SEQ ID No:6 and SEQ ID No:7 is provided in Table 2 under SEQ ID No:9. The sequence of the LRR domain of the beta-WOLF 0 allele amplified by primers having SEQ ID No:7and SEQ ID No:8 is provided in Table 2 under SEQ ID No:11.

Finally, the obtained sequences were translated into the corresponding amino acid

sequence of the LRR domain having SEQ ID No:10 and SEQ ID No:12 for the alpha-WOLF 15 allele and the beta-WOLF 0, respectively (See also Table 2). If PCR products were to be sequenced using SMRT sequencing (Pacific Biosciences),

PCR primers and PCR conditions were different.

To the above-mentioned forward primers the following standard amplification

sequence was added: GCAGTCGAACATGTAGCTGACTCAGGTCAC. To the reverse primer, the following standard amplification sequence was added:

TGGATCACTTGTGCAAGCATCACATCGTAG.

EXAMPLE3 Introducing alpha-WOLF 15 allele in a plant not carrying the allele

A spinach plant comprising the alpha-WOLF 15 allele, of which a representative

sample of seed was deposited with the NCIMB under NCIMB accession number 42466 was

crossed with a plant of variety Viroflay carrying the beta-WOLF 0 allele to obtain a F1 generation.

Subsequently, a F1 plant was selfed to obtain a F2 population.

Plants of the F2 population were assayed as described in Example 1 for resistance to

Peronosporafarinosaf. sp. spinaciaepfs:15. Approximately 75% of the plants scored completely

resistant in the assay.

Genomic DNA of each plant of the same F2 population was isolated and used in two

different polymerase chain reactions (PCR). The first PCR reaction was done using primers for

amplifying the LRR domain of an alpha-WOLF allele and the second PCR reaction was done using

primers for amplifying the LRR domain of a beta-WOLF allele, both as described in Example 2. The PCR products were visualized on agarose gel (not shown), this demonstrated that

approximately 25% of the plant only contained an alpha-WOLF fragment, approximately 50%

contained both an alpha- and a beta-WOLF fragment, and that the remaining approximately 25%

of the plants only contained a beta-WOLF fragment. The plants containing the alpha-WOLF

fragment completely correlated with the plants that scored resistant for pfs:15. The plants only

comprising the beta-WOLF fragment completely correlated with the plants that scored susceptible

for pfs:15. DNA from the PCR reaction was purified, and subsequently the sequence of the PCR

products was determined. The alpha-WOLF PCR products gave a sequence that corresponded to

the sequence of SEQ ID No:9, the genomic sequence of the LRR domain of the alpha-WOLF 15

allele. The beta-WOLF PCR products gave a sequence that corresponded to the sequence of SEQ

ID No: Ithe genomic sequence of the LRR domain of the beta-WOLF 0 allele.

Page 1 of 21 SEQUENCE LISTING

<110> Rijk Zwaan Zaadteelt en Zaadhandel B.V.

<120> PERONOSPORA RESISTANCE IN SPINACIA OLERACEA

<130> L/2VX23/KK/410p

<140>

<160> 12

<170> BiSSAP 1.2

<210> 1 <211> 6853 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..6853 <223> /organism="Spinacia oleracea" /mol_type="unassigned DNA"

<400> 1 atggccgaaa tcggatactc ggtttgtgcg aaactcatcg aagtgattgg cagtgagctg 60

atcaaagaga tttgtgacac atggggttac aaatctcttc ttgaggacct caacaaaact 120

gtattgacgg tcaggaacgt tctcattcaa gccggggtga tgcgggagct tactagtgaa 180

caacaaggtt tcattgcaga ccttaaagat gttgtttatg atgctgatga cttgttcgac 240

aagttactca ctcgtgctga gcgaaaacag attgatggaa acgaaatctc tgaaaaggta 300

cgtcgtttct tttcctctag taacaagatc ggtcaagctt actacatgtc tcgtaaggtt 360

aaggaaatta agaagcagtt ggatgaaatt gttgataggc atacaaaatt tgggtttagt 420

gccgagttta tacctgtttg tagggaaagg gggaacgaga gggaaacacg ttcatatata 480

gatgtcaaga atattcttgg gagggataaa gataagaatg atatcataga taggttgctt 540

aatcgtaatg gtaatgaage ttgtagtttc ctgaccatag tgggagcggg aggattggga 600

aaaactgctc ttgcacaact tgtgttcaat gatgaaaggg tcaaaattga gttccatgat 660

ttgaggtatt gggtttgtgt ctctgatcaa gatgggggcc aatttgatgt gaaagaaato 720

ctttgtaaga ttttagaggt ggttactaag gagaaagttg ataatagtto cacattggaa 780

ttggtacaaa gccaatttca agagaagtta agaggaaaga agtacttect tgttcttgat 840

gatgtatgga acgaagatcg tgagaagtgg cttcctttgg aagagttgtt aatgttgggt 900

caagggggaa gcaaggttgt agtgaccgca cgttcagaga agacagcaaa tgtcataggg 960

aaaagacatt tttatacact ggaatgtttg tcaccagatt attcatggag cttatttgaa 1020

atgtcggctt ttcagaaagg gcatgagcag gaaaaccatc acgaactagt tgatattggg 1080

aaaaagattg ttgaaaaatg ttataacaat ccacttgcta taacggtggt aggaagtctt 1140

Page 2 of 21

ctttatggag aggagataag taagtggcgg tcatttgaaa tgagtgagtt ggccaaaatt 1200

ggcaatgggg ataataagat tttgccgata ttaaagctca gttaccataa tcttataccc 1260

tcgttgaaga gttgcttcag ttattgtgca gtgtttccca aggatcatga aataaagaag 1320

gagatgttga ttgatctttg gatagcacaa ggatacgttg tggcacttga tggaggtcaa 1380

agtatagaag atgctgccga agaacatttt gtaattttgt tacggagatg tttctttcaa 1440

gatgtaaaga aggatgaata tggtgatgtt gattctgtta aaatccacga cttgatgcac 1500

gatgtcgccc aagaagtggg gagggaggaa atatgtgtag tgaatgataa tacaaagaac 1560

ttgggtgata aaatccgtca tgtacatggt gatgtcaata gatatgcaca aagagtctct 1620

ctgtgtagcc atagccataa gattcgttcg tatattggtg gtgattgtga aaaacgttgt 1680

gtggatacac taatagacaa gtggatgtgt cttaggatgt tggacttgtc atggtcggat 1740

gttaaaaatt tgcctaattc aataggtaaa ttgttgcact tgaggtatct taacctgtca 1800

gataatagaa atctaaagat acttcctgat gcaattacaa gactgcataa tttgcagaca 1860

ctgcttttag aagattgcag aagtttaaag gagttgccaa aagatttttg caaattggtc 1920

aaactgaggc acttggaatt acagggttgt catgatttga ttggtatgcc atttggaatg 1980

gataagctaa ctagtcttag aatactacca aacattgtgg tgggtaggaa ggaacaaagt 2040

gatgatgage tgaaagccct aaaaggcctc accgagataa aaggctccat ttctatcaga 2100

atctattcaa agtatagaat agttgaaggc atgaatgaca caggaggage tgcttatttg 2160

aagagcatga aacatctcag ggagattgat attacatttt tgggtgaatg tgttggccct 2220

gaagctgtat tggaaacctt agagccacct tcaaatatca agagcttata tatatataat 2280

tacagtggta caacaattcc agtatgggga agagcagaga ttaattgggc aatctccctc 2340

tcacatctcg tcgacatcca gcttagttgt tgtagtaatt tgcaggagat gccagtgctg 2400

agtaaactgc ctcatttgaa atcgctgaaa cttggatggt tggataactt agagtacatg 2460

gagagtagca gtagcagtga cacagaagca gcaacaccag aattaccaac attcttccct 2520

tcccttgaaa aacttacttt acagcatctg gaaaagttga agggttttgg gaacaggaga 2580

tcgagtagtt ttccccgcct ctctgaattg gaaatcaaga aatgcccaga tctaacgtca 2640

tttccttctt gtccaagcct tgagaagttg gaattgaaag aaagcaatga agcattgcaa 2700

ataatagtaa aaataacaac aagaggtaaa gaaaaagaag agaacaataa tgctggtgtt 2760

agaaattcac aagatgatga caaagtcaaa ttacggaaga tggtgataga caatctgggt 2820

tatctcacgg gggttgatat tagatttgat gatagagaag gtggatttgt taaccctgaa 2880

gctgtgttgg caaccctaga gccaccttca aatatcaaga gcttatctat acatcgtttt 2940

gatggtaaaa cacttccagt atggggaaga gcagagatta attgggcaat ctccctctca 3000

Page 3 of 21 catcttgtcg acatccagct ttggcattgt cgtaatttgc aggagatgcc agtgctgagt 3060

aaactgcctc atttgaaatc actggaactt tataatttga ttagtttaga gtacatggag 3120

agcacaagca gaagcagtag cagtgacaca gaagcagcaa caccagaatt accaacattc 3180

ttcccttccc ttgaaaaact tagactttgg tatctggaaa agttgaaggg tttggggaac 3240

aggagaccga gtagttttcc ccgcctctct gaattggaaa tctgggaatg cccagatcta 3300

acgtggtttc ctccttgtcc aagccttaaa acgttgaaat tggaaaaaaa caatgaagcg 3360

ttgcaaataa tagtaaaaat aacaacaaca agaggtaaag aagaaaaaga agaagacaag 3420

aatgctggtg ttggaaattc acaagatgat gacaatgtca aattacggaa ggtggaaata 3480

gacaatgtga gttatctcaa atcactgccc acaaattgtc ttactcacct caaaataact 3540

ggaatagatt acagggaggg ggagattgaa tcagattccg tggaggagga gattgaattg 3600

gaagttgggg aggcatttca gaagtgtgca tcttctttga gaagcctcat cataatcgga 3660

aatcacggaa taaataaagt gatgagactg tctggaagaa cagggttgga gcatttcact 3720

ctgttggact cactcaaatt ttcaaagata gaagaccagg aagatgaggg cgaagacaac 3780

atcatattct ggaaatcctt tcctcaaaac cttcgcagtt tgagaattaa agactctgac 3840

aaaatgacaa gtttgcccat ggggatgcag tacttaacct ccctccaaac cctcgaacta 3900

tcatattgtg atgaattgaa ttcccttcca gaatggataa gcagcttatc atctcttcaa 3960

tacctgcgca tatactactg tccagccctg aaatcactac cagaagcaat gcggaacctc 4020

acctcccttc agacacttgg gatatcggat tgtccagacc tagttaaaag atgcagaaaa 4080

cccaaccggca aggactatcc caaaattcaa cacatcccca aaattgtaag tcattgcaga 4140

aagtaattta ttcatttata tttattttat gcttagaatg atatacgcag tcgtcctttg 4200

gtttcaaatc ttgaatttgg tttttgtttt ctttctttgt ttctttattc aacaccagcc 4260

catttatgat tgattcatta aaaaaaggat ggagttttat ggatttgaag aagacaacga 4320

attgagattc ctggggtttt ctttttgttg gggttggatt tcatgtatat gttgctgatt 4380

aaatacgaga ctgatgatga tgatgtgttt atgggtttta aatcagatta aatatatggg 4440

aaatgcaagt taatttggga tgcacataag gtgtttgctg aaatgtctat gagaaatgtt 4500

gtttcttgga cttagaatga tatacactgt cgtcctttgg tttccaatct tacatttggt 4560

ttgtgttttc ttagtttgtt tctttaatca acaccaaccc gtttttttta aactacctgc 4620

aactactaat ttacgtttac cctgtatctc aggtactaaa tgaatattgg tgattttcag 4680

ttactcaaca ctagcttgat cctgaacgca cccaaccttc aggttagaat ccggcttact 4740

catccttttg tccagttttc aagtaattgt tttggcagga tcaattctct aattgttgta 4800

caccgtatat tgcaatttat agtgactaca gttaatgaat gtttacaaaa aattagtcat 4860

gtaaaaactt cttctctgtc cattacataa actctttttc tctttctaac ttatcatgtt 4920

Page 4 of 21

catgtctaaa caattaaaca tgctcacatc aatgttcatt taagctaact tacttctgta 4980

agagagcgag ctagttaaaa actcctttaa ctttctgttt tatactcagg acatggattg 5040

atgcaagcat gaagaacttc gggaatttgc taaaactcta ccaaagcgat gagagtttgg 5100

actttatttc acttgaagtc agggactgtc aacaaagcca cagtgtgcat gttggctgtt 5160

tcacttggac gataaaaagg tttatttaat tgttttccta agtgtatttg gcttacaagc 5220

ttttactttt cacttgaaag ggtttttctt gttttaagct tttcgaatta gagttttcgg 5280

ttgaagtaag agtagtcgta ttagtctttt acctaaggaa gactcttttt tgtaattttc 5340

agactatgca attcaagttt tcgagtgttt tcttgcttgt gtgattgtga gttggtgaat 5400

tcgtctttca tacattttga gattatcaga agctttatgc tccaccggta gtctagtacc 5460

ttttctgtta ctgtgcaggg aagtaatctg gtaccttcta tatatatgga aaaacataca 5520

ttatacatta tgcaaaattc ttacaggtta gttacttcct ggaacttcat ttacacttag 5580

ttttttttgt tccattccct cggaatcaag tcattccctc tgagaaatat gtaatgaact 5640

tctgtatgtt gctgtttggt tcctgtttta atcttcaatt ttcttgtata gttacagctg 5700

catttacaat gaagtttaag cagacactct ctttatatag tgcctctttc tggagcaccg 5760

tagagctgtc tgtggttgat caccatctgc tgccgagaga ttcagcaatc gcgtgtttga 5820

tcaggtaaaa gtttttatgt caatgtgttt ttttttccgt ttgatcaatt tatgtctgta 5880

ttcagattct tatcttctta cagtagcata acacattgtt tctttcattt atgtaaactg 5940

tttcaagatt acagagatgt atgcttcagt cgacattgat gataacttaa gatggcattc 6000

ctacaacagt tgcaggcgca ttctaactcc ggcaattcta gttaggcaag aggagcattg 6060

ccaatacctg ccacctctgg gatttactat accagggttg aagtttatgg aagacaccag 6120

ctatgcacaa gccttcaagg ggtcatccta cataacaagt tgaaccaacc aattgcttgt 6180

tggttcagtg gtaattgaag ctgaatttgg tagggatggc ccgtgttcga tccccacaac 6240

aacaattggg aggggactgg aacctatcca cacagaactc gccctgaatc cggattagcc 6300

ctaagggtga acggggtgct aacaccaaaa aaaaaaacat aacaagttga accaaacata 6360

ctttgtttga attgaagatt tagtgatttc atttgatcga ttgagatgtc ttattataag 6420

cgtatatgct cttggatttg gccacttagg tgttgtttga caattggaca ttaactcgct 6480

tttatatttt cttttctctt aggaaaggtg atcctgagaa tttatattgg aacacttttt 6540

ttttctcact agctttaaaa aagtgttctg tgttacctgc aattcaattt gattattttt 6600

cacatagttt tacctgaaaa agtgttacct gaaaaagtgt tacctgaaaa tcaactgaca 6660

taagtttttg tttggatcca attaaggaca ctagataaat cggaataaat aatcaaccaa 6720

ttaagtactt cataattaaa tatgaagtgt attattatct tatgcttgtg acattgaagg 6780

Page 5 of 21 atgttatgat attttaactc aataccttgc aaaatatact ggttaaattt cttaacaagg 6840

taacttggca aca 6853

<210> 2 <211> 4146 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..4146 <223> /organism="Spinacia oleracea" /mol_type="unassigned DNA"

<400> 2 atggccgaaa tcggatactc ggtttgtgcg aaactcatcg aagtgattgg cagtgagctg 60

atcaaagaga tttgtgacac atggggttac aaatctcttc ttgaggacct caacaaaact 120

gtattgacgg tcaggaacgt tctcattcaa gccggggtga tgcgggagct tactagtgaa 180

caacaaggtt tcattgcaga ccttaaagat gttgtttatg atgctgatga cttgttcgac 240

aagttactca ctcgtgctga gcgaaaacag attgatggaa acgaaatctc tgaaaaggta 300

cgtcgtttct tttcctctag taacaagatc ggtcaagctt actacatgtc tcgtaaggtt 360

aaggaaatta agaagcagtt ggatgaaatt gttgataggc atacaaaatt tgggtttagt 420

gccgagttta tacctgtttg tagggaaagg gggaacgaga gggaaacacg ttcatatata 480

gatgtcaaga atattcttgg gagggataaa gataagaatg atatcataga taggttgctt 540

aatcgtaatg gtaatgaage ttgtagtttc ctgaccatag tgggagcggg aggattggga 600

aaaactgctc ttgcacaact tgtgttcaat gatgaaaggg tcaaaattga gttccatgat 660

ttgaggtatt gggtttgtgt ctctgatcaa gatgggggcc aatttgatgt gaaagaaatc 720

ctttgtaaga ttttagaggt ggttactaag gagaaagttg ataatagttc cacattggaa 780

ttggtacaaa gccaatttca agagaagtta agaggaaaga agtacttect tgttcttgat 840

gatgtatgga acgaagatcg tgagaagtgg cttcctttgg aagagttgtt aatgttgggt 900

caagggggaa gcaaggttgt agtgaccgca cgttcagaga agacagcaaa tgtcataggg 960

aaaagacatt tttatacact ggaatgtttg tcaccagatt attcatggag cttatttgaa 1020

atgtcggctt ttcagaaagg gcatgagcag gaaaaccatc acgaactagt tgatattggg 1080

aaaaagattg ttgaaaaatg ttataacaat ccacttgcta taacggtggt aggaagtctt 1140

ctttatggag aggagataag taagtggcgg tcatttgaaa tgagtgagtt ggccaaaatt 1200

ggcaatgggg ataataagat tttgccgata ttaaagctca gttaccataa tcttataccc 1260

tcgttgaaga gttgcttcag ttattgtgca gtgtttccca aggatcatga aataaagaag 1320

gagatgttga ttgatctttg gatagcacaa ggatacgttg tggcacttga tggaggtcaa 1380

Page 6 of 21 agtatagaag atgctgccga agaacatttt gtaattttgt tacggagatg tttctttcaa 1440

gatgtaaaga aggatgaata tggtgatgtt gattctgtta aaatccacga cttgatgcac 1500

gatgtcgccc aagaagtggg gagggaggaa atatgtgtag tgaatgataa tacaaagaac 1560

ttgggtgata aaatccgtca tgtacatggt gatgtcaata gatatgcaca aagagtctct 1620

ctgtgtagcc atagccataa gattcgttcg tatattggtg gtgattgtga aaaacgttgt 1680

gtggatacac taatagacaa gtggatgtgt cttaggatgt tggacttgtc atggtcggat 1740

gttaaaaatt tgcctaattc aataggtaaa ttgttgcact tgaggtatct taacctgtca 1800

gataatagaa atctaaagat acttcctgat gcaattacaa gactgcataa tttgcagaca 1860

ctgcttttag aagattgcag aagtttaaag gagttgccaa aagatttttg caaattggtc 1920

aaactgaggc acttggaatt acagggttgt catgatttga ttggtatgcc atttggaatg 1980

gataagctaa ctagtcttag aatactacca aacattgtgg tgggtaggaa ggaacaaagt 2040

gatgatgage tgaaagccct aaaaggcctc accgagataa aaggctccat ttctatcaga 2100

atctattcaa agtatagaat agttgaaggc atgaatgaca caggaggage tgcttatttg 2160

aagagcatga aacatctcag ggagattgat attacatttt tgggtgaatg tgttggccct 2220

gaagctgtat tggaaacctt agagccacct tcaaatatca agagcttata tatatataat 2280

tacagtggta caacaattcc agtatgggga agagcagaga ttaattgggc aatctccctc 2340

tcacatctcg tcgacatcca gcttagttgt tgtagtaatt tgcaggagat gccagtgctg 2400

agtaaactgc ctcatttgaa atcgctgaaa cttggatggt tggataactt agagtacatg 2460

gagagtagca gtagcagtga cacagaagca gcaacaccag aattaccaac attcttccct 2520

tcccttgaaa aacttacttt acagcatctg gaaaagttga agggttttgg gaacaggaga 2580

tcgagtagtt ttccccgcct ctctgaattg gaaatcaaga aatgcccaga tctaacgtca 2640

tttccttctt gtccaagcct tgagaagttg gaattgaaag aaagcaatga agcattgcaa 2700

ataatagtaa aaataacaac aagaggtaaa gaaaaagaag agaacaataa tgctggtgtt 2760

agaaattcac aagatgatga caaagtcaaa ttacggaaga tggtgataga caatctgggt 2820

tatctcacgg gggttgatat tagatttgat gatagagaag gtggatttgt taaccctgaa 2880

gctgtgttgg caaccctaga gccaccttca aatatcaaga gcttatctat acatcgtttt 2940

gatggtaaaa cacttccagt atggggaaga gcagagatta attgggcaat ctccctctca 3000

catcttgtcg acatccagct ttggcattgt cgtaatttgc aggagatgcc agtgctgagt 3060

aaactgcctc atttgaaatc actggaactt tataatttga ttagtttaga gtacatggag 3120

agcacaagca gaagcagtag cagtgacaca gaagcagcaa caccagaatt accaacattc 3180

ttcccttccc ttgaaaaact tagactttgg tatctggaaa agttgaaggg tttggggaac 3240

aggagaccga gtagttttcc ccgcctctct gaattggaaa tctgggaatg cccagatcta 3300

Page 7 of 21

acgtggtttc ctccttgtcc aagccttaaa acgttgaaat tggaaaaaaa caatgaagcg 3360

ttgcaaataa tagtaaaaat aacaacaaca agaggtaaag aagaaaaaga agaagacaag 3420

aatgctggtg ttggaaattc acaagatgat gacaatgtca aattacggaa ggtggaaata 3480

gacaatgtga gttatctcaa atcactgccc acaaattgtc ttactcacct caaaataact 3540

ggaatagatt acagggaggg ggagattgaa tcagattccg tggaggagga gattgaattg 3600

gaagttgggg aggcatttca gaagtgtgca tcttctttga gaagcctcat cataatcgga 3660

aatcacggaa taaataaagt gatgagactg tctggaagaa cagggttgga gcatttcact 3720

ctgttggact cactcaaatt ttcaaagata gaagaccagg aagatgaggg cgaagacaac 3780

atcatattct ggaaatcctt tcctcaaaac cttcgcagtt tgagaattaa agactctgac 3840

aaaatgacaa gtttgcccat ggggatgcag tacttaacct ccctccaaac cctcgaacta 3900

tcatattgtg atgaattgaa ttcccttcca gaatggataa gcagcttatc atctcttcaa 3960

tacctgcgca tatactactg tccagccctg aaatcactac cagaagcaat gcggaacctc 4020

acctcccttc agacacttgg gatatcggat tgtccagacc tagttaaaag atgcagaaaa 4080

cccaaccggca aggactatcc caaaattcaa cacatcccca aaattgtact aaatgaatat 4140

tggtga 4146

<210> 3 <211> 4179 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..4179 <223> /organism="Spinacia oleracea" /mol_type="unassigned DNA"

<400> 3 atggccgaaa tcggatactc ggtttgtgcg aaactcatcg aagtgattgg cagtgagctg 60

atcaaagaga tttgtgacac atggggttac aaatctcttc ttgaggacct caacaaaact 120

gtattgacgg tcaggaacgt tctcattcaa gccggggtga tgcgggagct tactagtgaa 180

caacaaggtt tcattgcaga ccttaaagat gttgtttatg atgctgatga cttgttcgac 240

aagttactca ctcgtgctga gcgaaaacag attgatggaa acgaaatctc tgaaaaggta 300

cgtcgtttct tttcctctag taacaagatc ggtcaagctt actacatgtc tcgtaaggtt 360

aaggaaatta agaagcagtt ggatgaaatt gttgataggc atacaaaatt tgggtttagt 420

gccgagttta tacctgtttg tagggaaagg gggaacgaga gggaaacacg ttcatatata 480

gatgtcaaga atattcttgg gagggataaa gataagaatg atatcataga taggttgctt 540

aatcgtaatg gtaatgaage ttgtagtttc ctgaccatag tgggagcggg aggattggga 600

Page 8 of 21

aaaactgctc ttgcacaact tgtgttcaat gatgaaaggg tcaaaattga gttccatgat 660

ttgaggtatt gggtttgtgt ctctgatcaa gatgggggcc aatttgatgt gaaagaaatc 720

ctttgtaaga ttttagaggt ggttactaag gagaaagttg ataatagttc cacattggaa 780

ttggtacaaa gccaatttca agagaagtta agaggaaaga agtacttect tgttcttgat 840

gatgtatgga acgaagatcg tgagaagtgg cttcctttgg aagagttgtt aatgttgggt 900

caagggggaa gcaaggttgt agtgaccgca cgttcagaga agacagcaaa tgtcataggg 960

aaaagacatt tttatacact ggaatgtttg tcaccagatt attcatggag cttatttgaa 1020

atgtcggctt ttcagaaagg gcatgagcag gaaaaccatc acgaactagt tgatattggg 1080

aaaaagattg ttgaaaaatg ttataacaat ccacttgcta taacggtggt aggaagtctt 1140

ctttatggag aggagataag taagtggcgg tcatttgaaa tgagtgagtt ggccaaaatt 1200

ggcaatgggg ataataagat tttgccgata ttaaagctca gttaccataa tcttataccc 1260

tcgttgaaga gttgcttcag ttattgtgca gtgtttccca aggatcatga aataaagaag 1320

gagatgttga ttgatctttg gatagcacaa ggatacgttg tggcacttga tggaggtcaa 1380

agtatagaag atgctgccga agaacatttt gtaattttgt tacggagatg tttctttcaa 1440

gatgtaaaga aggatgaata tggtgatgtt gattctgtta aaatccacga cttgatgcac 1500

gatgtcgccc aagaagtggg gagggaggaa atatgtgtag tgaatgataa tacaaagaac 1560

ttgggtgata aaatccgtca tgtacatggt gatgtcaata gatatgcaca aagagtctct 1620

ctgtgtagcc atagccataa gattcgttcg tatattggtg gtgattgtga aaaacgttgt 1680

gtggatacac taatagacaa gtggatgtgt cttaggatgt tggacttgtc atggtcggat 1740

gttaaaaatt tgcctaattc aataggtaaa ttgttgcact tgaggtatct taacctgtca 1800

gataatagaa atctaaagat acttcctgat gcaattacaa gactgcataa tttgcagaca 1860

ctgcttttag aagattgcag aagtttaaag gagttgccaa aagatttttg caaattggtc 1920

aaactgaggc acttggaatt acagggttgt catgatttga ttggtatgcc atttggaatg 1980

gataagctaa ctagtcttag aatactacca aacattgtgg tgggtaggaa ggaacaaagt 2040

gatgatgage tgaaagccct aaaaggcctc accgagataa aaggctccat ttctatcaga 2100

atctattcaa agtatagaat agttgaaggc atgaatgaca caggaggage tgcttatttg 2160

aagagcatga aacatctcag ggagattgat attacatttt tgggtgaatg tgttggccct 2220

gaagctgtat tggaaacctt agagccacct tcaaatatca agagcttata tatatataat 2280

tacagtggta caacaattcc agtatgggga agagcagaga ttaattgggc aatctccctc 2340

tcacatctcg tcgacatcca gcttagttgt tgtagtaatt tgcaggagat gccagtgctg 2400

agtaaactgc ctcatttgaa atcgctgaaa cttggatggt tggataactt agagtacatg 2460

Page 9 of 21 gagagtagca gtagcagtga cacagaagca gcaacaccag aattaccaac attcttccct 2520

tcccttgaaa aacttacttt acagcatctg gaaaagttga agggttttgg gaacaggaga 2580

tcgagtagtt ttccccgcct ctctgaattg gaaatcaaga aatgcccaga tctaacgtca 2640

tttccttctt gtccaagcct tgagaagttg gaattgaaag aaagcaatga agcattgcaa 2700

ataatagtaa aaataacaac aagaggtaaa gaaaaagaag agaacaataa tgctggtgtt 2760

agaaattcac aagatgatga caaagtcaaa ttacggaaga tggtgataga caatctgggt 2820

tatctcacgg gggttgatat tagatttgat gatagagaag gtggatttgt taaccctgaa 2880

gctgtgttgg caaccctaga gccaccttca aatatcaaga gcttatctat acatcgtttt 2940

gatggtaaaa cacttccagt atggggaaga gcagagatta attgggcaat ctccctctca 3000

catcttgtcg acatccagct ttggcattgt cgtaatttgc aggagatgcc agtgctgagt 3060

aaactgcctc atttgaaatc actggaactt tataatttga ttagtttaga gtacatggag 3120

agcacaagca gaagcagtag cagtgacaca gaagcagcaa caccagaatt accaacattc 3180

ttcccttccc ttgaaaaact tagactttgg tatctggaaa agttgaaggg tttggggaac 3240

aggagaccga gtagttttcc ccgcctctct gaattggaaa tctgggaatg cccagatcta 3300

acgtggtttc ctccttgtcc aagccttaaa acgttgaaat tggaaaaaaa caatgaagcg 3360

ttgcaaataa tagtaaaaat aacaacaaca agaggtaaag aagaaaaaga agaagacaag 3420

aatgctggtg ttggaaattc acaagatgat gacaatgtca aattacggaa ggtggaaata 3480

gacaatgtga gttatctcaa atcactgccc acaaattgtc ttactcacct caaaataact 3540

ggaatagatt acagggaggg ggagattgaa tcagattccg tggaggagga gattgaattg 3600

gaagttgggg aggcatttca gaagtgtgca tcttctttga gaagcctcat cataatcgga 3660

aatcacggaa taaataaagt gatgagactg tctggaagaa cagggttgga gcatttcact 3720

ctgttggact cactcaaatt ttcaaagata gaagaccagg aagatgaggg cgaagacaac 3780

atcatattct ggaaatcctt tcctcaaaac cttcgcagtt tgagaattaa agactctgac 3840

aaaatgacaa gtttgcccat ggggatgcag tacttaacct ccctccaaac cctcgaacta 3900

tcatattgtg atgaattgaa ttcccttcca gaatggataa gcagcttatc atctcttcaa 3960

tacctgcgca tatactactg tccagccctg aaatcactac cagaagcaat gcggaacctc 4020

acctcccttc agacacttgg gatatcggat tgtccagacc tagttaaaag atgcagaaaa 4080

cccaaccggca aggactatcc caaaattcaa cacatcccca aaattttact caacactage 4140

ttgatcctga acgcacccaa ccttcaggac atggattga 4179

<210> 4 <211> 1381 <212> PRT <213> Spinacia oleracea

Page 10 of 21

<400> 4 Met Ala Glu Ile Gly Tyr Ser Val Cys Ala Lys Leu Ile Glu Val Ile 1 5 10 15 Gly Ser Glu Leu Ile Lys Glu Ile Cys Asp Thr Trp Gly Tyr Lys Ser 20 25 30 Leu Leu Glu Asp Leu Asn Lys Thr Val Leu Thr Val Arg Asn Val Leu 35 40 45 Ile Gln Ala Gly Val Met Arg Glu Leu Thr Ser Glu Gln Gln Gly Phe 50 55 60 Ile Ala Asp Leu Lys Asp Val Val Tyr Asp Ala Asp Asp Leu Phe Asp 70 75 80 Lys Leu Leu Thr Arg Ala Glu Arg Lys Gln Ile Asp Gly Asn Glu Ile 85 90 95 Ser Glu Lys Val Arg Arg Phe Phe Ser Ser Ser Asn Lys Ile Gly Gln 100 105 110 Ala Tyr Tyr Met Ser Arg Lys Val Lys Glu Ile Lys Lys Gln Leu Asp 115 120 125 Glu Ile Val Asp Arg His Thr Lys Phe Gly Phe Ser Ala Glu Phe Ile 130 135 140 Pro Val Cys Arg Glu Arg Gly Asn Glu Arg Glu Thr Arg Ser Tyr Ile 145 150 155 160 Asp Val Lys Asn Ile Leu Gly Arg Asp Lys Asp Lys Asn Asp Ile Ile 165 170 175 Asp Arg Leu Leu Asn Arg Asn Gly Asn Glu Ala Cys Ser Phe Leu Thr 180 185 190 Ile Val Gly Ala Gly Gly Leu Gly Lys Thr Ala Leu Ala Gln Leu Val 195 200 205 Phe Asn Asp Glu Arg Val Lys Ile Glu Phe His Asp Leu Arg Tyr Trp 210 215 220 Val Cys Val Ser Asp Gln Asp Gly Gly Gln Phe Asp Val Lys Glu Ile 225 230 235 240 Leu Cys Lys Ile Leu Glu Val Val Thr Lys Glu Lys Val Asp Asn Ser 245 250 255 Ser Thr Leu Glu Leu Val Gln Ser Gln Phe Gln Glu Lys Leu Arg Gly 260 265 270 Lys Lys Tyr Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Arg Glu 275 280 285 Lys Trp Leu Pro Leu Glu Glu Leu Leu Met Leu Gly Gln Gly Gly Ser 290 295 300 Lys Val Val Val Thr Ala Arg Ser Glu Lys Thr Ala Asn Val Ile Gly 305 310 315 320 Lys Arg His Phe Tyr Thr Leu Glu Cys Leu Ser Pro Asp Tyr Ser Trp 325 330 335 Ser Leu Phe Glu Met Ser Ala Phe Gln Lys Gly His Glu Gln Glu Asn 340 345 350 His His Glu Leu Val Asp Ile Gly Lys Lys Ile Val Glu Lys Cys Tyr 355 360 365 Asn Asn Pro Leu Ala Ile Thr Val Val Gly Ser Leu Leu Tyr Gly Glu 370 375 380 Glu Ile Ser Lys Trp Arg Ser Phe Glu Met Ser Glu Leu Ala Lys Ile 385 390 395 400 Gly Asn Gly Asp Asn Lys Ile Leu Pro Ile Leu Lys Leu Ser Tyr His 405 410 415 Asn Leu Ile Pro Ser Leu Lys Ser Cys Phe Ser Tyr Cys Ala Val Phe 420 425 430 Pro Lys Asp His Glu Ile Lys Lys Glu Met Leu Ile Asp Leu Trp Ile 435 440 445 Ala Gln Gly Tyr Val Val Ala Leu Asp Gly Gly Gln Ser Ile Glu Asp 450 455 460 Ala Ala Glu Glu His Phe Val Ile Leu Leu Arg Arg Cys Phe Phe Gln 465 470 475 480

Page 11 of 21 Asp Val Lys Lys Asp Glu Tyr Gly Asp Val Asp Ser Val Lys Ile His 485 490 495 Asp Leu Met His Asp Val Ala Gln Glu Val Gly Arg Glu Glu Ile Cys 500 505 510 Val Val Asn Asp Asn Thr Lys Asn Leu Gly Asp Lys Ile Arg His Val 515 520 525 His Gly Asp Val Asn Arg Tyr Ala Gln Arg Val Ser Leu Cys Ser His 530 535 540 Ser His Lys Ile Arg Ser Tyr Ile Gly Gly Asp Cys Glu Lys Arg Cys 545 550 555 560 Val Asp Thr Leu Ile Asp Lys Trp Met Cys Leu Arg Met Leu Asp Leu 565 570 575 Ser Trp Ser Asp Val Lys Asn Leu Pro Asn Ser Ile Gly Lys Leu Leu 580 585 590 His Leu Arg Tyr Leu Asn Leu Ser Asp Asn Arg Asn Leu Lys Ile Leu 595 600 605 Pro Asp Ala Ile Thr Arg Leu His Asn Leu Gln Thr Leu Leu Leu Glu 610 615 620 Asp Cys Arg Ser Leu Lys Glu Leu Pro Lys Asp Phe Cys Lys Leu Val 625 630 635 640 Lys Leu Arg His Leu Glu Leu Gln Gly Cys His Asp Leu Ile Gly Met 645 650 655 Pro Phe Gly Met Asp Lys Leu Thr Ser Leu Arg Ile Leu Pro Asn Ile 660 665 670 Val Val Gly Arg Lys Glu Gln Ser Asp Asp Glu Leu Lys Ala Leu Lys 675 680 685 Gly Leu Thr Glu Ile Lys Gly Ser Ile Ser Ile Arg Ile Tyr Ser Lys 690 695 700 Tyr Arg Ile Val Glu Gly Met Asn Asp Thr Gly Gly Ala Ala Tyr Leu 705 710 715 720 Lys Ser Met Lys His Leu Arg Glu Ile Asp Ile Thr Phe Leu Gly Glu 725 730 735 Cys Val Gly Pro Glu Ala Val Leu Glu Thr Leu Glu Pro Pro Ser Asn 740 745 750 Ile Lys Ser Leu Tyr Ile Tyr Asn Tyr Ser Gly Thr Thr Ile Pro Val 755 760 765 Trp Gly Arg Ala Glu Ile Asn Trp Ala Ile Ser Leu Ser His Leu Val 770 775 780 Asp Ile Gln Leu Ser Cys Cys Ser Asn Leu Gln Glu Met Pro Val Leu 785 790 795 800 Ser Lys Leu Pro His Leu Lys Ser Leu Lys Leu Gly Trp Leu Asp Asn 805 810 815 Leu Glu Tyr Met Glu Ser Ser Ser Ser Ser Asp Thr Glu Ala Ala Thr 820 825 830 Pro Glu Leu Pro Thr Phe Phe Pro Ser Leu Glu Lys Leu Thr Leu Gln 835 840 845 His Leu Glu Lys Leu Lys Gly Phe Gly Asn Arg Arg Ser Ser Ser Phe 850 855 860 Pro Arg Leu Ser Glu Leu Glu Ile Lys Lys Cys Pro Asp Leu Thr Ser 865 870 875 880 Phe Pro Ser Cys Pro Ser Leu Glu Lys Leu Glu Leu Lys Glu Ser Asn 885 890 895 Glu Ala Leu Gln Ile Ile Val Lys Ile Thr Thr Arg Gly Lys Glu Lys 900 905 910 Glu Glu Asn Asn Asn Ala Gly Val Arg Asn Ser Gln Asp Asp Asp Lys 915 920 925 Val Lys Leu Arg Lys Met Val Ile Asp Asn Leu Gly Tyr Leu Thr Gly 930 935 940 Val Asp Ile Arg Phe Asp Asp Arg Glu Gly Gly Phe Val Asn Pro Glu 945 950 955 960 Ala Val Leu Ala Thr Leu Glu Pro Pro Ser Asn Ile Lys Ser Leu Ser 965 970 975 Ile His Arg Phe Asp Gly Lys Thr Leu Pro Val Trp Gly Arg Ala Glu

Page 12 of 21 980 985 990 Ile Asn Trp Ala Ile Ser Leu Ser His Leu Val Asp Ile Gln Leu Trp 995 1000 1005 His Cys Arg Asn Leu Gln Glu Met Pro Val Leu Ser Lys Leu Pro His 1010 1015 1020 Leu Lys Ser Leu Glu Leu Tyr Asn Leu Ile Ser Leu Glu Tyr Met Glu 1025 1030 1035 1040 Ser Thr Ser Arg Ser Ser Ser Ser Asp Thr Glu Ala Ala Thr Pro Glu 1045 1050 1055 Leu Pro Thr Phe Phe Pro Ser Leu Glu Lys Leu Arg Leu Trp Tyr Leu 1060 1065 1070 Glu Lys Leu Lys Gly Leu Gly Asn Arg Arg Pro Ser Ser Phe Pro Arg 1075 1080 1085 Leu Ser Glu Leu Glu Ile Trp Glu Cys Pro Asp Leu Thr Trp Phe Pro 1090 1095 1100 Pro Cys Pro Ser Leu Lys Thr Leu Lys Leu Glu Lys Asn Asn Glu Ala 1105 1110 1115 1120 Leu Gln Ile Ile Val Lys Ile Thr Thr Thr Arg Gly Lys Glu Glu Lys 1125 1130 1135 Glu Glu Asp Lys Asn Ala Gly Val Gly Asn Ser Gln Asp Asp Asp Asn 1140 1145 1150 Val Lys Leu Arg Lys Val Glu Ile Asp Asn Val Ser Tyr Leu Lys Ser 1155 1160 1165 Leu Pro Thr Asn Cys Leu Thr His Leu Lys Ile Thr Gly Ile Asp Tyr 1170 1175 1180 Arg Glu Gly Glu Ile Glu Ser Asp Ser Val Glu Glu Glu Ile Glu Leu 1185 1190 1195 1200 Glu Val Gly Glu Ala Phe Gln Lys Cys Ala Ser Ser Leu Arg Ser Leu 1205 1210 1215 Ile Ile Ile Gly Asn His Gly Ile Asn Lys Val Met Arg Leu Ser Gly 1220 1225 1230 Arg Thr Gly Leu Glu His Phe Thr Leu Leu Asp Ser Leu Lys Phe Ser 1235 1240 1245 Lys Ile Glu Asp Gln Glu Asp Glu Gly Glu Asp Asn Ile Ile Phe Trp 1250 1255 1260 Lys Ser Phe Pro Gln Asn Leu Arg Ser Leu Arg Ile Lys Asp Ser Asp 1265 1270 1275 1280 Lys Met Thr Ser Leu Pro Met Gly Met Gln Tyr Leu Thr Ser Leu Gln 1285 1290 1295 Thr Leu Glu Leu Ser Tyr Cys Asp Glu Leu Asn Ser Leu Pro Glu Trp 1300 1305 1310 Ile Ser Ser Leu Ser Ser Leu Gln Tyr Leu Arg Ile Tyr Tyr Cys Pro 1315 1320 1325 Ala Leu Lys Ser Leu Pro Glu Ala Met Arg Asn Leu Thr Ser Leu Gln 1330 1335 1340 Thr Leu Gly Ile Ser Asp Cys Pro Asp Leu Val Lys Arg Cys Arg Lys 1345 1350 1355 1360 Pro Asn Gly Lys Asp Tyr Pro Lys Ile Gln His Ile Pro Lys Ile Val 1365 1370 1375 Leu Asn Glu Tyr Trp 1380

<210> 5 <211> 1392 <212> PRT <213> Spinacia oleracea

<400> 5 Met Ala Glu Ile Gly Tyr Ser Val Cys Ala Lys Leu Ile Glu Val Ile 1 5 10 15 Gly Ser Glu Leu Ile Lys Glu Ile Cys Asp Thr Trp Gly Tyr Lys Ser 20 25 30

Page 13 of 21 Leu Leu Glu Asp Leu Asn Lys Thr Val Leu Thr Val Arg Asn Val Leu 35 40 45 Ile Gln Ala Gly Val Met Arg Glu Leu Thr Ser Glu Gln Gln Gly Phe 50 55 60 Ile Ala Asp Leu Lys Asp Val Val Tyr Asp Ala Asp Asp Leu Phe Asp 70 75 80 Lys Leu Leu Thr Arg Ala Glu Arg Lys Gln Ile Asp Gly Asn Glu Ile 85 90 95 Ser Glu Lys Val Arg Arg Phe Phe Ser Ser Ser Asn Lys Ile Gly Gln 100 105 110 Ala Tyr Tyr Met Ser Arg Lys Val Lys Glu Ile Lys Lys Gln Leu Asp 115 120 125 Glu Ile Val Asp Arg His Thr Lys Phe Gly Phe Ser Ala Glu Phe Ile 130 135 140 Pro Val Cys Arg Glu Arg Gly Asn Glu Arg Glu Thr Arg Ser Tyr Ile 145 150 155 160 Asp Val Lys Asn Ile Leu Gly Arg Asp Lys Asp Lys Asn Asp Ile Ile 165 170 175 Asp Arg Leu Leu Asn Arg Asn Gly Asn Glu Ala Cys Ser Phe Leu Thr 180 185 190 Ile Val Gly Ala Gly Gly Leu Gly Lys Thr Ala Leu Ala Gln Leu Val 195 200 205 Phe Asn Asp Glu Arg Val Lys Ile Glu Phe His Asp Leu Arg Tyr Trp 210 215 220 Val Cys Val Ser Asp Gln Asp Gly Gly Gln Phe Asp Val Lys Glu Ile 225 230 235 240 Leu Cys Lys Ile Leu Glu Val Val Thr Lys Glu Lys Val Asp Asn Ser 245 250 255 Ser Thr Leu Glu Leu Val Gln Ser Gln Phe Gln Glu Lys Leu Arg Gly 260 265 270 Lys Lys Tyr Phe Leu Val Leu Asp Asp Val Trp Asn Glu Asp Arg Glu 275 280 285 Lys Trp Leu Pro Leu Glu Glu Leu Leu Met Leu Gly Gln Gly Gly Ser 290 295 300 Lys Val Val Val Thr Ala Arg Ser Glu Lys Thr Ala Asn Val Ile Gly 305 310 315 320 Lys Arg His Phe Tyr Thr Leu Glu Cys Leu Ser Pro Asp Tyr Ser Trp 325 330 335 Ser Leu Phe Glu Met Ser Ala Phe Gln Lys Gly His Glu Gln Glu Asn 340 345 350 His His Glu Leu Val Asp Ile Gly Lys Lys Ile Val Glu Lys Cys Tyr 355 360 365 Asn Asn Pro Leu Ala Ile Thr Val Val Gly Ser Leu Leu Tyr Gly Glu 370 375 380 Glu Ile Ser Lys Trp Arg Ser Phe Glu Met Ser Glu Leu Ala Lys Ile 385 390 395 400 Gly Asn Gly Asp Asn Lys Ile Leu Pro Ile Leu Lys Leu Ser Tyr His 405 410 415 Asn Leu Ile Pro Ser Leu Lys Ser Cys Phe Ser Tyr Cys Ala Val Phe 420 425 430 Pro Lys Asp His Glu Ile Lys Lys Glu Met Leu Ile Asp Leu Trp Ile 435 440 445 Ala Gln Gly Tyr Val Val Ala Leu Asp Gly Gly Gln Ser Ile Glu Asp 450 455 460 Ala Ala Glu Glu His Phe Val Ile Leu Leu Arg Arg Cys Phe Phe Gln 465 470 475 480 Asp Val Lys Lys Asp Glu Tyr Gly Asp Val Asp Ser Val Lys Ile His 485 490 495 Asp Leu Met His Asp Val Ala Gln Glu Val Gly Arg Glu Glu Ile Cys 500 505 510 Val Val Asn Asp Asn Thr Lys Asn Leu Gly Asp Lys Ile Arg His Val 515 520 525 His Gly Asp Val Asn Arg Tyr Ala Gln Arg Val Ser Leu Cys Ser His

Page 14 of 21 530 535 540 Ser His Lys Ile Arg Ser Tyr Ile Gly Gly Asp Cys Glu Lys Arg Cys 545 550 555 560 Val Asp Thr Leu Ile Asp Lys Trp Met Cys Leu Arg Met Leu Asp Leu 565 570 575 Ser Trp Ser Asp Val Lys Asn Leu Pro Asn Ser Ile Gly Lys Leu Leu 580 585 590 His Leu Arg Tyr Leu Asn Leu Ser Asp Asn Arg Asn Leu Lys Ile Leu 595 600 605 Pro Asp Ala Ile Thr Arg Leu His Asn Leu Gln Thr Leu Leu Leu Glu 610 615 620 Asp Cys Arg Ser Leu Lys Glu Leu Pro Lys Asp Phe Cys Lys Leu Val 625 630 635 640 Lys Leu Arg His Leu Glu Leu Gln Gly Cys His Asp Leu Ile Gly Met 645 650 655 Pro Phe Gly Met Asp Lys Leu Thr Ser Leu Arg Ile Leu Pro Asn Ile 660 665 670 Val Val Gly Arg Lys Glu Gln Ser Asp Asp Glu Leu Lys Ala Leu Lys 675 680 685 Gly Leu Thr Glu Ile Lys Gly Ser Ile Ser Ile Arg Ile Tyr Ser Lys 690 695 700 Tyr Arg Ile Val Glu Gly Met Asn Asp Thr Gly Gly Ala Ala Tyr Leu 705 710 715 720 Lys Ser Met Lys His Leu Arg Glu Ile Asp Ile Thr Phe Leu Gly Glu 725 730 735 Cys Val Gly Pro Glu Ala Val Leu Glu Thr Leu Glu Pro Pro Ser Asn 740 745 750 Ile Lys Ser Leu Tyr Ile Tyr Asn Tyr Ser Gly Thr Thr Ile Pro Val 755 760 765 Trp Gly Arg Ala Glu Ile Asn Trp Ala Ile Ser Leu Ser His Leu Val 770 775 780 Asp Ile Gln Leu Ser Cys Cys Ser Asn Leu Gln Glu Met Pro Val Leu 785 790 795 800 Ser Lys Leu Pro His Leu Lys Ser Leu Lys Leu Gly Trp Leu Asp Asn 805 810 815 Leu Glu Tyr Met Glu Ser Ser Ser Ser Ser Asp Thr Glu Ala Ala Thr 820 825 830 Pro Glu Leu Pro Thr Phe Phe Pro Ser Leu Glu Lys Leu Thr Leu Gln 835 840 845 His Leu Glu Lys Leu Lys Gly Phe Gly Asn Arg Arg Ser Ser Ser Phe 850 855 860 Pro Arg Leu Ser Glu Leu Glu Ile Lys Lys Cys Pro Asp Leu Thr Ser 865 870 875 880 Phe Pro Ser Cys Pro Ser Leu Glu Lys Leu Glu Leu Lys Glu Ser Asn 885 890 895 Glu Ala Leu Gln Ile Ile Val Lys Ile Thr Thr Arg Gly Lys Glu Lys 900 905 910 Glu Glu Asn Asn Asn Ala Gly Val Arg Asn Ser Gln Asp Asp Asp Lys 915 920 925 Val Lys Leu Arg Lys Met Val Ile Asp Asn Leu Gly Tyr Leu Thr Gly 930 935 940 Val Asp Ile Arg Phe Asp Asp Arg Glu Gly Gly Phe Val Asn Pro Glu 945 950 955 960 Ala Val Leu Ala Thr Leu Glu Pro Pro Ser Asn Ile Lys Ser Leu Ser 965 970 975 Ile His Arg Phe Asp Gly Lys Thr Leu Pro Val Trp Gly Arg Ala Glu 980 985 990 Ile Asn Trp Ala Ile Ser Leu Ser His Leu Val Asp Ile Gln Leu Trp 995 1000 1005 His Cys Arg Asn Leu Gln Glu Met Pro Val Leu Ser Lys Leu Pro His 1010 1015 1020 Leu Lys Ser Leu Glu Leu Tyr Asn Leu Ile Ser Leu Glu Tyr Met Glu 1025 1030 1035 1040

Page 15 of 21 Ser Thr Ser Arg Ser Ser Ser Ser Asp Thr Glu Ala Ala Thr Pro Glu 1045 1050 1055 Leu Pro Thr Phe Phe Pro Ser Leu Glu Lys Leu Arg Leu Trp Tyr Leu 1060 1065 1070 Glu Lys Leu Lys Gly Leu Gly Asn Arg Arg Pro Ser Ser Phe Pro Arg 1075 1080 1085 Leu Ser Glu Leu Glu Ile Trp Glu Cys Pro Asp Leu Thr Trp Phe Pro 1090 1095 1100 Pro Cys Pro Ser Leu Lys Thr Leu Lys Leu Glu Lys Asn Asn Glu Ala 1105 1110 1115 1120 Leu Gln Ile Ile Val Lys Ile Thr Thr Thr Arg Gly Lys Glu Glu Lys 1125 1130 1135 Glu Glu Asp Lys Asn Ala Gly Val Gly Asn Ser Gln Asp Asp Asp Asn 1140 1145 1150 Val Lys Leu Arg Lys Val Glu Ile Asp Asn Val Ser Tyr Leu Lys Ser 1155 1160 1165 Leu Pro Thr Asn Cys Leu Thr His Leu Lys Ile Thr Gly Ile Asp Tyr 1170 1175 1180 Arg Glu Gly Glu Ile Glu Ser Asp Ser Val Glu Glu Glu Ile Glu Leu 1185 1190 1195 1200 Glu Val Gly Glu Ala Phe Gln Lys Cys Ala Ser Ser Leu Arg Ser Leu 1205 1210 1215 Ile Ile Ile Gly Asn His Gly Ile Asn Lys Val Met Arg Leu Ser Gly 1220 1225 1230 Arg Thr Gly Leu Glu His Phe Thr Leu Leu Asp Ser Leu Lys Phe Ser 1235 1240 1245 Lys Ile Glu Asp Gln Glu Asp Glu Gly Glu Asp Asn Ile Ile Phe Trp 1250 1255 1260 Lys Ser Phe Pro Gln Asn Leu Arg Ser Leu Arg Ile Lys Asp Ser Asp 1265 1270 1275 1280 Lys Met Thr Ser Leu Pro Met Gly Met Gln Tyr Leu Thr Ser Leu Gln 1285 1290 1295 Thr Leu Glu Leu Ser Tyr Cys Asp Glu Leu Asn Ser Leu Pro Glu Trp 1300 1305 1310 Ile Ser Ser Leu Ser Ser Leu Gln Tyr Leu Arg Ile Tyr Tyr Cys Pro 1315 1320 1325 Ala Leu Lys Ser Leu Pro Glu Ala Met Arg Asn Leu Thr Ser Leu Gln 1330 1335 1340 Thr Leu Gly Ile Ser Asp Cys Pro Asp Leu Val Lys Arg Cys Arg Lys 1345 1350 1355 1360 Pro Asn Gly Lys Asp Tyr Pro Lys Ile Gln His Ile Pro Lys Ile Leu 1365 1370 1375 Leu Asn Thr Ser Leu Ile Leu Asn Ala Pro Asn Leu Gln Asp Met Asp 1380 1385 1390

<210> 6 <211> 20 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..20 <223> /organism="Spinacia oleracea" /note="Primer" /mol_type="unassigned DNA"

<400> 6 acaagtggat gtgtcttagg 20

Page 16 of 21 <210> 7 <211> 19 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..19 <223> /organism="Spinacia oleracea" /note="Primer" /mol_type="unassigned DNA"

<400> 7 ttcgccctca tcttcctgg 19

<210> 8 <211> 18 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..18 <223> /organism="Spinacia oleracea" /note="Primer" /mol__type="unassigned DNA"

<400> 8 tcacgtgggt tgtgttgt 18

<210> 9 <211> 2078 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1..2078 <223> /organism="Spinacia oleracea" /mol_type="unassigned DNA"

<400> 9 acaagtggat gtgtcttagg atgttggact tgtcatggtc ggatgttaaa aatttgccta 60

attcaatagg taaattgttg cacttgaggt atcttaacct gtcagataat agaaatctaa 120

agatacttcc tgatgcaatt acaagactgc ataatttgca gacactgctt ttagaagatt 180

gcagaagttt aaaggagttg ccaaaagatt tttgcaaatt ggtcaaactg aggcacttgg 240

aattacaggg ttgtcatgat ttgattggta tgccatttgg aatggataag ctaactagtc 300

ttagaatact accaaacatt gtggtgggta ggaaggaaca aagtgatgat gagctgaaag 360

ccctaaaagg cctcaccgag ataaaaggct ccatttctat cagaatctat tcaaagtata 420

gaatagttga aggcatgaat gacacaggag gagctgctta tttgaagage atgaaacatc 480

tcagggagat tgatattaca tttttgggtg aatgtgttgg ccctgaagct gtattggaaa 540

ccttagagcc accttcaaat atcaagagct tatatatata taattacagt ggtacaacaa 600

Page 17 of 21

ttccagtatg gggaagagca gagattaatt gggcaatctc cctctcacat ctcgtcgaca 660

tccagcttag ttgttgtagt aatttgcagg agatgccagt gctgagtaaa ctgcctcatt 720

tgaaatcgct gaaacttgga tggttggata acttagagta catggagagt agcagtagca 780

gtgacacaga agcagcaaca ccagaattac caacattctt cccttccctt gaaaaactta 840

ctttacagca tctggaaaag ttgaagggtt ttgggaacag gagatcgagt agttttcccc 900

gcctctctga attggaaatc aagaaatgcc cagatctaac gtcatttcct tcttgtccaa 960

gccttgagaa gttggaattg aaagaaagca atgaagcatt gcaaataata gtaaaaataa 1020

caacaagagg taaagaaaaa gaagagaaca ataatgctgg tgttagaaat tcacaagatg 1080

atgacaaagt caaattacgg aagatggtga tagacaatct gggttatctc acgggggttg 1140

atattagatt tgatgataga gaaggtggat ttgttaaccc tgaagctgtg ttggcaaccc 1200

tagagccacc ttcaaatatc aagagcttat ctatacatcg ttttgatggt aaaacacttc 1260

cagtatgggg aagagcagag attaattggg caatctccct ctcacatctt gtcgacatcc 1320

agctttggca ttgtcgtaat ttgcaggaga tgccagtgct gagtaaactg cctcatttga 1380

aatcactgga actttataat ttgattagtt tagagtacat ggagagcaca agcagaagca 1440

gtagcagtga cacagaagca gcaacaccag aattaccaac attcttccct tcccttgaaa 1500

aacttagact ttggtatctg gaaaagttga agggtttggg gaacaggaga ccgagtagtt 1560

ttccccgcct ctctgaattg gaaatctggg aatgcccaga tctaacgtgg tttcctcctt 1620

gtccaagcct taaaacgttg aaattggaaa aaaacaatga agcgttgcaa ataatagtaa 1680

aaataacaac aacaagaggt aaagaagaaa aagaagaaga caagaatgct ggtgttggaa 1740

attcacaaga tgatgacaat gtcaaattac ggaaggtgga aatagacaat gtgagttatc 1800

tcaaatcact gcccacaaat tgtcttactc acctcaaaat aactggaata gattacaggg 1860

agggggagat tgaatcagat tccgtggagg aggagattga attggaagtt ggggaggcat 1920

ttcagaagtg tgcatcttct ttgagaagcc tcatcataat cggaaatcac ggaataaata 1980

aagtgatgag actgtctgga agaacagggt tggagcattt cactctgttg gactcactca 2040

aattttcaaa gatagaagac caggaagatg agggcgaa 2078

<210> 10 <211> 692 <212> PRT <213> Spinacia oleracea

<400> 10 Lys Trp Met Cys Leu Arg Met Leu Asp Leu Ser Trp Ser Asp Val Lys 1 5 10 15 Asn Leu Pro Asn Ser Ile Gly Lys Leu Leu His Leu Arg Tyr Leu Asn 20 25 30

Page 18 of 21 Leu Ser Asp Asn Arg Asn Leu Lys Ile Leu Pro Asp Ala Ile Thr Arg 35 40 45 Leu His Asn Leu Gln Thr Leu Leu Leu Glu Asp Cys Arg Ser Leu Lys 50 55 60 Glu Leu Pro Lys Asp Phe Cys Lys Leu Val Lys Leu Arg His Leu Glu 70 75 80 Leu Gln Gly Cys His Asp Leu Ile Gly Met Pro Phe Gly Met Asp Lys 85 90 95 Leu Thr Ser Leu Arg Ile Leu Pro Asn Ile Val Val Gly Arg Lys Glu 100 105 110 Gln Ser Asp Asp Glu Leu Lys Ala Leu Lys Gly Leu Thr Glu Ile Lys 115 120 125 Gly Ser Ile Ser Ile Arg Ile Tyr Ser Lys Tyr Arg Ile Val Glu Gly 130 135 140 Met Asn Asp Thr Gly Gly Ala Ala Tyr Leu Lys Ser Met Lys His Leu 145 150 155 160 Arg Glu Ile Asp Ile Thr Phe Leu Gly Glu Cys Val Gly Pro Glu Ala 165 170 175 Val Leu Glu Thr Leu Glu Pro Pro Ser Asn Ile Lys Ser Leu Tyr Ile 180 185 190 Tyr Asn Tyr Ser Gly Thr Thr Ile Pro Val Trp Gly Arg Ala Glu Ile 195 200 205 Asn Trp Ala Ile Ser Leu Ser His Leu Val Asp Ile Gln Leu Ser Cys 210 215 220 Cys Ser Asn Leu Gln Glu Met Pro Val Leu Ser Lys Leu Pro His Leu 225 230 235 240 Lys Ser Leu Lys Leu Gly Trp Leu Asp Asn Leu Glu Tyr Met Glu Ser 245 250 255 Ser Ser Ser Ser Asp Thr Glu Ala Ala Thr Pro Glu Leu Pro Thr Phe 260 265 270 Phe Pro Ser Leu Glu Lys Leu Thr Leu Gln His Leu Glu Lys Leu Lys 275 280 285 Gly Phe Gly Asn Arg Arg Ser Ser Ser Phe Pro Arg Leu Ser Glu Leu 290 295 300 Glu Ile Lys Lys Cys Pro Asp Leu Thr Ser Phe Pro Ser Cys Pro Ser 305 310 315 320 Leu Glu Lys Leu Glu Leu Lys Glu Ser Asn Glu Ala Leu Gln Ile Ile 325 330 335 Val Lys Ile Thr Thr Arg Gly Lys Glu Lys Glu Glu Asn Asn Asn Ala 340 345 350 Gly Val Arg Asn Ser Gln Asp Asp Asp Lys Val Lys Leu Arg Lys Met 355 360 365 Val Ile Asp Asn Leu Gly Tyr Leu Thr Gly Val Asp Ile Arg Phe Asp 370 375 380 Asp Arg Glu Gly Gly Phe Val Asn Pro Glu Ala Val Leu Ala Thr Leu 385 390 395 400 Glu Pro Pro Ser Asn Ile Lys Ser Leu Ser Ile His Arg Phe Asp Gly 405 410 415 Lys Thr Leu Pro Val Trp Gly Arg Ala Glu Ile Asn Trp Ala Ile Ser 420 425 430 Leu Ser His Leu Val Asp Ile Gln Leu Trp His Cys Arg Asn Leu Gln 435 440 445 Glu Met Pro Val Leu Ser Lys Leu Pro His Leu Lys Ser Leu Glu Leu 450 455 460 Tyr Asn Leu Ile Ser Leu Glu Tyr Met Glu Ser Thr Ser Arg Ser Ser 465 470 475 480 Ser Ser Asp Thr Glu Ala Ala Thr Pro Glu Leu Pro Thr Phe Phe Pro 485 490 495 Ser Leu Glu Lys Leu Arg Leu Trp Tyr Leu Glu Lys Leu Lys Gly Leu 500 505 510 Gly Asn Arg Arg Pro Ser Ser Phe Pro Arg Leu Ser Glu Leu Glu Ile 515 520 525 Trp Glu Cys Pro Asp Leu Thr Trp Phe Pro Pro Cys Pro Ser Leu Lys

Page 19 of 21 530 535 540 Thr Leu Lys Leu Glu Lys Asn Asn Glu Ala Leu Gln Ile Ile Val Lys 545 550 555 560 Ile Thr Thr Thr Arg Gly Lys Glu Glu Lys Glu Glu Asp Lys Asn Ala 565 570 575 Gly Val Gly Asn Ser Gln Asp Asp Asp Asn Val Lys Leu Arg Lys Val 580 585 590 Glu Ile Asp Asn Val Ser Tyr Leu Lys Ser Leu Pro Thr Asn Cys Leu 595 600 605 Thr His Leu Lys Ile Thr Gly Ile Asp Tyr Arg Glu Gly Glu Ile Glu 610 615 620 Ser Asp Ser Val Glu Glu Glu Ile Glu Leu Glu Val Gly Glu Ala Phe 625 630 635 640 Gln Lys Cys Ala Ser Ser Leu Arg Ser Leu Ile Ile Ile Gly Asn His 645 650 655 Gly Ile Asn Lys Val Met Arg Leu Ser Gly Arg Thr Gly Leu Glu His 660 665 670 Phe Thr Leu Leu Asp Ser Leu Lys Phe Ser Lys Ile Glu Asp Gln Glu 675 680 685 Asp Glu Gly Glu 690

<210> 11 <211> 1597 <212> DNA <213> Spinacia oleracea

<220> <221> source <222> 1. 1597 <223> /organism="Spinacia oleracea" /mol_type="unassigned DNA"

<400> 11 tcacgtgggt tgtgttgtcg atagagatcc agaaatagtc tttttatgta gcaataagat 60

tcgttcgtat attagcggtc gctgcataaa gaatccggtg gattcacaaa tagacaactg 120

gatgtgcctt agggtgttgg acttgtcaga ttcatgtgtt aaagatttgt ctgattcaat 180

aggtaagctg ctgcacttaa ggtatcttaa cctctcttct aatataaagt tggagataat 240

ccctgatgca attacaagac tgcataactt gcagacacta cttttagaag attgcagaag 300

tttaaaggag ttgccaaaag atttttgcaa attggtcaaa ctgaggcact tggaattaca 360

gggttgtcat gatttgattg gtatgtcatt tggaatggat aagctaacta gtcttagaat 420

actaccaaac attgtggtgg gtaggaagga acaaagtgtt gatgatgage tgaaagccct 480

aaaaggcctc accgagataa aaggctccat tgatatcaca atctattcaa aatatagaag 540

agttgaaggc atgaatggca caggaggagg agctgggtat ttgaagagca tgaaacatct 600

cacgggggtt aatattacat ttgatgaagg tggatgtgtt aaccctgaag ctgtgtattt 660

gaagagcatg aaacatctca cgagggttat tattatattt gattataaag gtggatgtgt 720

taaccctgaa gctgtgttgg caaccctaga gccaccttca aatatcaaga ggttagagat 780

gtggcattac agtggtacaa caattccagt atggggaaga gcagagatta attgggcaat 840

ctccctctca catcttgtcg acatcacgct tgaagattgt tacaatttgc aggagatgcc 900

Page 20 of 21

agtgctgagt aaactgcctc atttgaaatc actggaactt acagagttgg ataacttaga 960

gtacatggag agtagaagca gcagcagtag cagtgacaca gaagcagcaa caccagaatt 1020

accaacattc ttcccttccc ttgaaaaact tacactttgg cgtctggaca agttgaaggg 1080

ttttgggaac aggagatcga gtagttttcc ccgcctctct aaattggaaa tctggaaatg 1140

tccagatcta acgtcatttc cttcttgtcc aagccttgaa gagttggaat tgaaagaaaa 1200

caatgaagcg ttgcaaataa tagtaaaaat aacaacaaca agaggtaaag aagaaaaaga 1260

agaagacaag aatgctggtg ttggaaattc acaagatgat gacaatgtca aattatggaa 1320

ggtggaaata gacaatctgg gttatctcaa atcactgccc acaaattgtc tgactcacct 1380

cgaccttaca ataagtgatt ccaaggaggg ggagggtgaa tgggaagttg gggatgcatt 1440

tcagaagtgt gtatcttctt tgagaagcct caccataatc ggaaatcacg gaataaataa 1500

agtgaagaga ctgtctggaa gaacagggtt ggagcatttc actctgttgg aatcactcaa 1560

actttcagat atagaagacc aggaagatga gggcgaa 1597

<210> 12 <211> 532 <212> PRT <213> Spinacia oleracea

<400> 12 His Val Gly Cys Val Val Asp Arg Asp Pro Glu Ile Val Phe Leu Cys 1 5 10 15 Ser Asn Lys Ile Arg Ser Tyr Ile Ser Gly Arg Cys Ile Lys Asn Pro 20 25 30 Val Asp Ser Gln Ile Asp Asn Trp Met Cys Leu Arg Val Leu Asp Leu 35 40 45 Ser Asp Ser Cys Val Lys Asp Leu Ser Asp Ser Ile Gly Lys Leu Leu 50 55 60 His Leu Arg Tyr Leu Asn Leu Ser Ser Asn Ile Lys Leu Glu Ile Ile 70 75 80 Pro Asp Ala Ile Thr Arg Leu His Asn Leu Gln Thr Leu Leu Leu Glu 85 90 95 Asp Cys Arg Ser Leu Lys Glu Leu Pro Lys Asp Phe Cys Lys Leu Val 100 105 110 Lys Leu Arg His Leu Glu Leu Gln Gly Cys His Asp Leu Ile Gly Met 115 120 125 Ser Phe Gly Met Asp Lys Leu Thr Ser Leu Arg Ile Leu Pro Asn Ile 130 135 140 Val Val Gly Arg Lys Glu Gln Ser Val Asp Asp Glu Leu Lys Ala Leu 145 150 155 160 Lys Gly Leu Thr Glu Ile Lys Gly Ser Ile Asp Ile Thr Ile Tyr Ser 165 170 175 Lys Tyr Arg Arg Val Glu Gly Met Asn Gly Thr Gly Gly Gly Ala Gly 180 185 190 Tyr Leu Lys Ser Met Lys His Leu Thr Gly Val Asn Ile Thr Phe Asp 195 200 205 Glu Gly Gly Cys Val Asn Pro Glu Ala Val Tyr Leu Lys Ser Met Lys 210 215 220 His Leu Thr Arg Val Ile Ile Ile Phe Asp Tyr Lys Gly Gly Cys Val 225 230 235 240

Page 21 of 21 Asn Pro Glu Ala Val Leu Ala Thr Leu Glu Pro Pro Ser Asn Ile Lys 245 250 255 Arg Leu Glu Met Trp His Tyr Ser Gly Thr Thr Ile Pro Val Trp Gly 260 265 270 Arg Ala Glu Ile Asn Trp Ala Ile Ser Leu Ser His Leu Val Asp Ile 275 280 285 Thr Leu Glu Asp Cys Tyr Asn Leu Gln Glu Met Pro Val Leu Ser Lys 290 295 300 Leu Pro His Leu Lys Ser Leu Glu Leu Thr Glu Leu Asp Asn Leu Glu 305 310 315 320 Tyr Met Glu Ser Arg Ser Ser Ser Ser Ser Ser Asp Thr Glu Ala Ala 325 330 335 Thr Pro Glu Leu Pro Thr Phe Phe Pro Ser Leu Glu Lys Leu Thr Leu 340 345 350 Trp Arg Leu Asp Lys Leu Lys Gly Phe Gly Asn Arg Arg Ser Ser Ser 355 360 365 Phe Pro Arg Leu Ser Lys Leu Glu Ile Trp Lys Cys Pro Asp Leu Thr 370 375 380 Ser Phe Pro Ser Cys Pro Ser Leu Glu Glu Leu Glu Leu Lys Glu Asn 385 390 395 400 Asn Glu Ala Leu Gln Ile Ile Val Lys Ile Thr Thr Thr Arg Gly Lys 405 410 415 Glu Glu Lys Glu Glu Asp Lys Asn Ala Gly Val Gly Asn Ser Gln Asp 420 425 430 Asp Asp Asn Val Lys Leu Trp Lys Val Glu Ile Asp Asn Leu Gly Tyr 435 440 445 Leu Lys Ser Leu Pro Thr Asn Cys Leu Thr His Leu Asp Leu Thr Ile 450 455 460 Ser Asp Ser Lys Glu Gly Glu Gly Glu Trp Glu Val Gly Asp Ala Phe 465 470 475 480 Gln Lys Cys Val Ser Ser Leu Arg Ser Leu Thr Ile Ile Gly Asn His 485 490 495 Gly Ile Asn Lys Val Lys Arg Leu Ser Gly Arg Thr Gly Leu Glu His 500 505 510 Phe Thr Leu Leu Glu Ser Leu Lys Leu Ser Asp Ile Glu Asp Gln Glu 515 520 525 Asp Glu Gly Glu

Claims (9)

1. Method for identifying or selecting a spinach plant carrying an allele designated alpha WOLF 15 which confers resistance to at least one Peronosporafarinosaf Sp. spinacea race when present in a spinach plant, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif "MAEIGYSVC" at its N-terminus; and b) the motif "KWMCLR"; and wherein the LRR domain of the protein has in order of increased preference at least 92%, 93%, 94%, 95%, 96%, 97%, 9899% , 100% sequence similarity to SEQ ID No:10, and wherein the allele when homozygously present in a spinach plant confers complete resistance to Peronosporafarinosaf. Sp. spinacea races pfs:1, pfs:2, pfs:3, pfs:4 and pfs: 5, pfs:6, pfs:8, pfs:9, pfs:11, pfs:12, pfs:13, pfs:14, pfs:15, pfs:16 and isolates UA1014 and US1508, and confers intermediate resistance to pfs:10, and does not confer resistance to pfs:7, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:1.

2. Method for identifying or selecting a spinach plant carrying the allele as defined in claim 1, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence similarity to SEQ ID No:2.

3. Method for identifying or selecting a spinach plant carrying the allele as defined in claim 1, comprising determining the presence of a genomic nucleotide sequence or a part thereof in the genome of a plant, wherein said sequence has in order of increased preference 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%sequence similarity to SEQ ID No:3.

4. The method as claimed in any one of the claims I to 3, comprising determining the presence of the LRR domain as defined in claim 1.

5. The method of claim 4, wherein the LRR domain is determined by using a primer pair to amplify the LRR domain, wherein the forward primer is a nucleic acid molecule having the sequence of SEQ ID No:6.

6.The method of claim 4, wherein the LRR domain is determined by using a primer pair to amplify the LRR domain, wherein the reverse primer is a nucleic acid molecule having the sequence of SEQ ID No:7.

7. Use of a primer pair comprising a forward primer which is a nucleic acid molecule having the sequence of SEQ ID No:6 and a reverse primer which is a nucleic acid molecule having the sequence of SEQ ID No:7.

8. A method for producing a spinach plant showing resistance to Peronosporafarinosaf. sp. spinaciae comprising: (a) crossing a plant comprising the allele as defined in claim 1, with another plant; (b) optionally performing one or more rounds of selfing and/or crossing; (c) selecting after one or more rounds of selfing and/or crossing for a plant that comprises said allele as defined in claim 1.

9. The method of claim 8, wherein the selection of a plant comprising the allele comprises determining the presence of the allele according to the method as claimed in any one of the claims 1 to 6.